CLINICAL MICROBIOLOGY REVIEWS, Oct. 2007, p. 550–578
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 20, No. 4
Clinical Significance of Human Immunodeficiency Virus Type 1
Carrie Dykes and Lisa M. Demeter*
Infectious Diseases Division, Department of Medicine, University of Rochester School of Medicine and
Dentistry, Rochester, New York
ASSAYS TO MEASURE HIV-1 REPLICATION FITNESS .................................................................................551
Cell Culture Assays ................................................................................................................................................551
General features of cell culture assays to measure HIV-1 replication fitness...........................................551
Growth competition assays versus parallel infections...................................................................................551
Single-cycle versus multiple-cycle assays.........................................................................................................554
Whole-virus versus recombinant-virus assays................................................................................................554
Direct measure of virus replication versus use of a reporter gene .............................................................555
Use of cell lines versus primary human cells.................................................................................................556
Other differences among fitness assays...........................................................................................................557
Examples of specific assays...............................................................................................................................557
Other Assays To Measure Fitness........................................................................................................................558
MATHEMATICAL APPROACHES TO QUANTIFYING REPLICATION FITNESS......................................558
Multiple-Cycle Assays ............................................................................................................................................558
EFFECTS OF SPECIFIC DRUG RESISTANCE MUTATIONS ON FITNESS................................................559
Mutations Conferring Resistance to Reverse Transcriptase Inhibitors .........................................................559
(ii) TAMs .........................................................................................................................................................561
(v) Q151M complex........................................................................................................................................561
Mutations Conferring Resistance to Protease Inhibitors.................................................................................562
Major protease inhibitor resistance mutations..............................................................................................563
Minor protease inhibitor resistance mutations..............................................................................................563
Mutations Conferring Resistance to Entry Inhibitors ......................................................................................564
MUTATIONAL INTERACTIONS THAT AFFECT FITNESS .............................................................................564
(i) Interactions among nRTI resistance mutations ...................................................................................564
(ii) Interactions between NNRTI and nRTI resistance mutations..........................................................564
(iii) Effects of codons in reverse transcriptase not associated with drug resistance on fitness
of nRTI-resistant mutants.........................................................................................................................565
Protease cleavage site mutations in gag ..........................................................................................................566
Insertions in gag..................................................................................................................................................566
Other mutations in gag......................................................................................................................................566
GENETIC DETERMINANTS OF FITNESS ..........................................................................................................567
CORRELATION OF HIV-1 REPLICATION FITNESS WITH CLINICAL OUTCOMES ..............................567
Transmission Efficiency .........................................................................................................................................568
Outcomes in Primary HIV-1 Infection.................................................................................................................568
Evolution of fitness during primary infection ................................................................................................568
Impact of HIV-1 replication fitness on CD4?T-cell count and viral load ................................................569
* Corresponding author. Mailing address: University of Rochester,
School of Medicine and Dentistry, Infectious Diseases Unit, 601 Elm-
wood Ave., Box 689, Rochester, NY 14642. Phone: (585) 275-4764.
Fax: (585) 442-9328. E-mail: firstname.lastname@example.org.
Outcomes in Chronic HIV-1 Infection.................................................................................................................570
Correlation of HIV-1 replication fitness with clinical outcome in untreated patients...................................570
Correlation between fitness and outcome of antiretroviral treatment interruptions................................571
Association of HIV-1 replication fitness with antiretroviral treatment responses....................................572
Clinical Predictive Potential of Specific HIV-1 Replication Fitness Assays...................................................572
Human immunodeficiency virus type 1 (HIV-1) is a retrovi-
rus that infects primarily CD4-expressing T cells. Primary
HIV-1 infection occurs with a single variant initially. However,
the rapid replication and turnover of HIV-1 (79, 177) and the
high mutation rate of the viral reverse transcriptase (110, 147)
result in high mutation frequencies that quickly lead to the
production of a population of genetically distinct but related
variants called a quasispecies (53). If the effective size of the
replicating HIV-1 population is large, the variant that predom-
inates in an HIV-1 quasispecies at any one point in time should
be the variant that is most fit under the selective pressures that
According to population genetics, fitness is defined as a
variant’s ability to contribute to successive generations (re-
viewed in reference 54). By this definition, HIV-1 variants with
high levels of fitness should have a selective advantage over
other less-fit variants in clinical infections. It has also been
postulated that HIV-1 variants with reduced fitness may be less
pathogenic, leading to improved clinical outcomes. HIV-1 fit-
ness has been studied primarily using in vitro cell culture
model systems in which the rate of replication is measured, and
it is not known how closely this measure of fitness correlates
with viral fitness in patients or clinical outcomes. For the sake
of simplicity, we will refer to all such assays as replication
fitness assays, recognizing that these assays are varied in their
design and do not fully reflect the selective forces impacting
viral fitness during clinical HIV-1 infection.
The degree to which HIV-1 replication fitness correlates
with clinical outcome continues to be a controversial issue. We
believe that this controversy stems in part from a lack of con-
sensus on what the best approaches are to measure fitness and
which clinical outcomes are most impacted by fitness. In addi-
tion, the labor-intensive nature of most assays used to quantify
fitness limits the sample sizes of studies correlating fitness with
clinical outcomes. This review will summarize and put into
perspective the types of assays used to measure HIV-1 repli-
cation fitness, the effects of specific drug resistance mutations
on fitness, and the studies done to date evaluating the corre-
lation between in vitro measures of HIV-1 replication fitness
and clinical outcome.
ASSAYS TO MEASURE HIV-1 REPLICATION FITNESS
Cell Culture Assays
General features of cell culture assays to measure HIV-1
replication fitness. The published literature contains data on a
multitude of cell culture assays that have been used to measure
HIV-1 replication fitness. This variety makes it difficult to
comprehensively list the different approaches to measuring
fitness and limits the comparisons that can be made between
studies from different groups of investigators. One common
feature of all assays is that they compare the replication of a
test variant to that of a reference strain. The test variant is
either a site-directed mutant of a laboratory strain of HIV-1 or
an isolate derived from a patient sample, usually peripheral
blood (Fig. 1a).
We have proposed further categorizing cell culture fitness
assays on the basis of five major features: whether replication
of the test strains and replication of the reference strains are
compared in parallel infections or directly in a single culture
(the latter is referred to as a growth competition assay);
whether replication is measured over a single virus life cycle,
using pseudotyped virus, or over multiple cycles; whether the
test virus is an isolate obtained from a clinical specimen
(“whole virus”) versus a recombinant virus containing only a
portion of the clinical viral sequence; whether virus is detected
directly by assaying a viral gene or protein or indirectly through
the use of a reporter gene; and whether the assay utilizes cell
lines versus primary human cells (Table 1). These features of
fitness assays are summarized in more detail below.
Growth competition assays versus parallel infections. Dif-
ferences between growth competition assays and parallel in-
fections are illustrated in Fig. 1b. Growth competition assays
are generally preferable to parallel infections for measuring
replication fitness since the replication of the test strain and
replication of the reference strain are compared in the same
culture, eliminating potential artifacts resulting from differ-
ences in culture conditions of the test and reference strains. As
the total virus population expands, the prevalence of the test
variant in a growth competition assay should decrease over
time relative to the reference strain if the test variant has lower
fitness. If the relative proportions of the test and reference
strains are compared at more than one time point after infec-
tion, growth competition assays are also relatively insensitive to
the specific method used to determine virus inoculum, which is
another significant advantage over parallel infections (more
detail on methods to quantify virus inoculum is provided be-
low). A third advantage of growth competition assays is that
they can detect small differences in replication fitness that are
not identified in parallel infections (33, 137).
An important potential disadvantage of growth competition
assays is that a quantitative assay that can distinguish the test
and reference strains must be available (Fig. 1b). Quantitation
of the relative amounts of test and reference strains usually
adds significantly to the complexity and cost of the assay. In
addition, the question of viral recombination must be consid-
ered, since the production of recombinant progeny viruses
could alter the apparent replication fitness of the test strain if
it differs genetically from the reference strain at more than one
nucleotide position. In order to minimize recombination be
VOL. 20, 2007 CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS551
FIG. 1. Approaches to measuring HIV-1 replication fitness in cell culture. (a) Production of a virus stock from the peripheral blood of an
HIV-infected patient. In this example, peripheral blood is obtained from the patient by venipuncture and separated into its component parts by
density gradient centrifugation. A whole-virus isolate is obtained by coculturing the patient’s PBMCs with a susceptible cell, either PBMCs from
an HIV-negative human donor or an appropriate cell line (left-hand side). The clinical isolate is illustrated as red hexagons and can be harvested
by separating the culture supernatant from the cells in culture. A recombinant virus derived from the patient’s HIV-1 strain can also be obtained
by purifying plasma and amplifying a specific region of the viral genome using reverse transcription followed by PCR (RT-PCR) (right-hand side).
The PCR product can then be cloned into a viral vector containing the remainder of the HIV genome. Recombinant virus (illustrated by red striped
hexagons) can then be produced by transfecting an appropriate cell line with the recombinant HIV vector. Either of these methods can be used
to generate the clinical test strain for a fitness assay, as illustrated in b. RBC, red blood cells. (b) Methods used to carry out growth competition assays versus
are illustrated by rectangles containing a circle, and nucleic acid is illustrated by curled lines. The reference virus, the cells that it infects, and the nucleic acid
derived from it are colored blue; the analogous illustrations for the test virus are colored red. Uninfected cells are white. In parallel infections (left-hand side of
measured by quantitating p24 capsid antigen or a reporter gene such as luciferase. In a growth competition assay, the reference and test viruses infect the same
the culture (A), and the relative amounts of each variant can be quantitated using real-time PCR, a heteroduplex tracking assay, or sequence analysis. More
recently, assays that utilize flow cytometry to detect reporter genes expressed by the viruses in infected cells have been developed (B). ELISA, enzyme-linked
tween the test and reference strains in a growth competition
assay, the experimental design should minimize dual infection
of cells, which is a prerequisite for retroviral recombination
(80), by using a low multiplicity of infection initially and lim-
iting the duration of infection.
Single-cycle versus multiple-cycle assays. Single-cycle assays
are typically conducted by deleting the envelope gene from an
HIV-1 vector and then producing pseudotyped virus by trans-
fecting an appropriate cell line with the env-deleted HIV vec-
tor together with a plasmid expressing a gene product that can
serve as an envelope (see Fig. 2 for a depiction of the Mono-
gram Biosciences replication capacity [RC] assay, which is a
single-cycle assay utilizing murine leukemia virus [MLV] env-
pseudotyped virus). The MLV envelope and vesicular stoma-
titis virus G protein have each been used to pseudotype HIV-1
variants in single-cycle replication fitness assays (15, 184). Such
pseudotyped virions can infect susceptible cells but cannot
produce infectious progeny due to the fact that the genome
still carries the env deletion. Infection with pseudotyped virus
is thus limited to a single round of replication. Single-cycle
infections have the advantage of a shorter time frame, typically
24 to 72 h, compared to several days to weeks for a multiple-
cycle assay. Multiple-cycle assays have the theoretical advan-
tage of greater sensitivity because differences between the two
variants can be amplified over many life cycles, although the
two types of assays have not been extensively compared.
Whole-virus versus recombinant-virus assays. Whole-virus
assays are performed using intact isolates cultured directly from
patient samples. In contrast, recombinant-virus assays require the
amplification of a region of interest by PCR, for example, pro-
tease and reverse transcriptase, and the subsequent cloning of
that region into an HIV-1 vector encoding the remaining viral
genome from a laboratory strain (Fig. 1a). The recombinant virus
may be derived from a single clone of the PCR amplicon, or a
pool of recombinant viruses can be produced from the bulk clon-
ing of all amplicons and transfection of the pooled viral vectors.
The major advantage of the recombinant-virus assay is that
it does not require the isolation of infectious HIV-1 from the
patient, which adds significantly to the time and cost of the
assay. In addition, viral vectors used in the recombinant-virus
assay can be modified to express a reporter gene, such as
luciferase, which can be used to detect viral infection. Recom-
binant assays allow one to look at the effects of a particular
gene segment on fitness but have the disadvantage of not
taking into account the possible modulating effects of other
gene segments located outside the region of amplification.
Whole-virus assays have the advantage of looking at the
replication of the complete viral population present in pe-
ripheral blood and thus are likely to be a more accurate
measure of viral fitness in a patient. However, whole-virus
assays are more difficult in that they require target cells that
can propagate all variant types contained within the isolate,
usually primary human peripheral blood mononuclear cells
(PBMCs); in addition, the methods to detect virus growth
are limited to those that directly assay viral genes or gene
Studies have compared whole-virus and recombinant-vi-
rus assays in relatively small numbers of clinical samples in
order to determine the relative impact of different gene
segments on overall replication. Using virus isolates without
drug resistance mutations in pol, overall fitness in a whole-
virus assay correlated most closely with that of a recombi-
nant-virus assay in which the env gene was amplified from
the patient specimen (142). In contrast, in one isolate with
TABLE 1. General features of cell culture assays to measure HIV-1 replication fitness
Parallel infection vs growth competition assay
Parallel infection................................................................................................................Test and reference viruses are grown in separate cultures
Growth competition assay................................................................................................Test and reference viruses are grown in the same culture
Single cycle vs multiple cycles of replication
Single cycle.........................................................................................................................Assays are conducted using an env-deleted viral vector that
completes only one replication cycle
Multiple cycles of replication...........................................................................................env is not deleted, and the virus completes several rounds
Whole virus vs recombinant virus
Whole virus ........................................................................................................................Assays are performed using intact isolates cultured from
Recombinant virus.............................................................................................................Assays are performed using a laboratory strain that
contains a portion of the patient’s viral genome
previously PCR amplified from a clinical sample
Direct measure of virus replication vs use of a reporter gene
Direct measure of virus replication.................................................................................Virus replication is measured by quantifying a viral protein
such as p24
Use of a reporter gene .....................................................................................................Virus replication is measured by quantifying a reporter
gene that is expressed by a recombinant virus such as
Use of cell lines vs primary human cells
Use of cell lines.................................................................................................................Assays are performed in a transformed T-cell line
Primary human cells..........................................................................................................Assays are performed in primary human cells (e.g.,
PBMCs or macrophages)
554DYKES AND DEMETERCLIN. MICROBIOL. REV.
drug resistance mutations, the pol gene appeared to make a
major contribution to viral fitness (142). In addition, studies
have demonstrated interactions among gag, protease, and
reverse transcriptase that affect viral fitness (19, 156). Thus,
the correlation between the two assay types will likely vary
and will depend on the gene segment included in the re-
combinant-virus assay, whether the patient population is
treatment-naive or -experienced, and, if treatment failure is
occurring, the duration of virologic failure.
Direct measure of virus replication versus use of a reporter
gene. Fitness assays can also vary in the methodologies used to
detect and quantify the test and reference strains (Fig. 1b). The
major distinguishing feature is whether an assay measures a viral
gene or gene product directly or whether a reporter gene is used
as a surrogate measure of viral replication. Some examples of
measuring virus directly include quantitation of a viral protein,
such as p24, by enzyme-linked immunosorbent assay (106, 156,
163); measurement of reverse transcriptase activity (164, 175);
FIG. 2. Design of the Monogram Biosciences RC assay. (a) Production of patient-derived recombinant viruses. HIV-1 genomic RNA is purified
from patient plasma. Reverse transcriptase PCR (RT-PCR) is used to amplify a region of the viral genome spanning the 3? end of gag, protease
(PR), and the first 313 codons of reverse transcriptase (RT). The pooled PCR amplicons are cloned into an HIV-1 vector containing a luciferase
reporter gene. The resulting recombinant HIV-1 clones are cotransfected together into a mammalian cell line with a plasmid that allows the
expression of an amphotropic MLV (A-MLV) envelope. The resultant pool of recombinant pseudotyped viruses will utilize the A-MLV envelope
to infect susceptible cells and will express patient-derived protease and reverse transcriptase as well as luciferase. The A-MLV envelope allows the
infection of CD4-negative cells. (b) Determination of the replication capacity of patient-derived recombinant viruses. The pool of recombinant
viruses is used to infect a cell line; virus replication is quantified by measuring luciferase activity at a single time point. Because the patient-derived
recombinant viruses do not encode an envelope protein, progeny viruses will not be infectious, i.e., only a single round of virus replication will
occur. (Reproduced from reference 15 with permission of the publisher.)
VOL. 20, 2007CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS 555
and quantitation of proviral DNA (116). In addition, in growth
competition assays, the relative amounts of test and reference
strains can be quantified by sequence analysis of proviral DNA or
viral RNA (65, 69, 78, 86, 117, 154, 155, 169, 174), heteroduplex
tracking assays (HTAs) (140), or allele-specific real-time PCR
(33, 142, 176). It should be noted that the latter methods do not
provide information on the extent of viral replication.
The most common method to directly quantify virus growth
is p24 antigen concentration in the culture supernatant. This
approach has been used to quantify the relative replication
rates of different variants in parallel infections (27, 163). p24
antigen concentration can also be used to evaluate the relative
expansion of virus during a growth competition experiment in
which the relative proportions of test and reference strains are
measured by a PCR-based method that does not directly mea-
sure virus replication (174).
A number of different methods have been used to quantify the
relative proportion of test and reference variants in a growth
competition assay. HTAs of the env gene have been used in
whole-virus growth competition assays (7, 116, 140, 164). Hybrid-
ization of a PCR-amplified product to a labeled nucleic acid
probe results in a heteroduplex that has a different electro-
phoretic mobility from that of the homoduplex if there are suffi-
cient sequence differences between the amplicon and the probe.
The mobility of the heteroduplex is affected more by insertions
and deletions than by base substitutions. Therefore, if the test and
reference strains are sufficiently divergent in their sequences,
HTAs can be used to quantify their relative proportions in a
growth competition assay over time. The env region is particularly
amenable to being assayed by HTA because it often exhibits
substantial sequence divergence and insertions/deletions among
HIV strains. This method is less labor-intensive than other meth-
ods that rely on PCR amplification of the test and reference
strains but requires that the two variants have substantial diver-
gence in their nucleotide sequences.
Sequence analysis has also been used to quantify the relative
amounts of the two variants in a growth competition assay
either by direct sequence analysis of the bulk PCR product (65,
69, 78, 86, 117, 154, 155, 174) or by analysis of individual clones
derived from the PCR amplicon (119). One disadvantage of
bulk sequencing is that differences in the surrounding se-
quence may influence the assay’s sensitivity for detecting a
minority variant at a given codon. This is less of a concern
when site-directed mutants that differ by only one or a few
codons are compared but may impact reproducibility when
clinical isolates are studied. In addition, quantitation of the
relative amounts of mutants by analysis of the sequencing
electropherogram from the bulk amplicon has a limited linear
range. Clonal sequence analysis does not have these limitations
if large numbers of clones are assayed, but this approach is
significantly more labor-intensive and costly.
Real-time PCR has also been used to quantify the relative
amounts of test and reference strains in a growth competition
assay by allele-specific amplification of viral nucleic acid at a
has an advantage over other molecular assays used to quantify the
relative proportion of the test and reference strains because of its
high throughput and wide linear range of detection. However,
surrounding genetic variation, which can influence the efficiency
of primer or probe hybridization to the target sequence, can
significantly influence the performance characteristics of allele-
specific real-time PCR. Therefore, if real-time PCR is used to
detect a specific viral mutation, the assay must be optimized and
validated for each codon of interest.
Examples of reporter genes used to detect viral replication are
luciferase (133), green fluorescent protein (GFP) (184), hisD
from Salmonella enterica serovar Typhimurium, human placental
alkaline phosphatase (PLAP) (103), and the mouse Thy1.1 and
Thy1.2 alleles (57). A major advantage of the enzymatic assay to
quantitation; a disadvantage is that different variants cannot be
distinguished, and therefore, a growth competition assay is not
feasible. The advantage of the paired reporter genes hisD/PLAP
cytometry, respectively, is that each gene in the pair can be cloned
distinguished in growth competition assays (57, 103). A further
potential advantage of the Thy1.1/Thy1.2 flow cytometry-based
assay is that the number of cells infected by each variant is quan-
tified, allowing an assessment of both virus growth and the rela-
tive proportions of test and reference strains (57). However, re-
porter genes are surrogate measures of viral replication, and
linkage of the HIV-1 genome and the reporter gene is necessary
for an accurate interpretation of the data; this linkage could
potentially be disrupted if recombination between the test and
reference strains that express different reporter genes were to
occur. This theoretical limitation has not posed a problem under
the conditions reported in previously published assays, which uti-
lized a low virus inoculum to initiate infection (57, 103).
Use of cell lines versus primary human cells. The specific
target cells used in fitness assays may also affect the apparent
fitness of a mutant. The best evidence for an impact of cell type
on fitness has been obtained for HIV-1 mutants that are resis-
tant to nucleoside analog inhibitors of the viral reverse trans-
criptase. For example, the replication fitness of M184V, which
is resistant to lamivudine, was reduced in primary human
PBMCs but was indistinguishable from that of wild-type HIV-1
in a lymphoid cell line (10). Purified reverse transcriptase with
the M184V mutation showed reduced processivity of polymer-
ization that was accentuated at low nucleotide concentrations
(10). Since PBMCs are known to have lower concentrations of
nucleotides than T-cell lines, this finding offers a potential
biochemical mechanism for the differences in fitness of this
mutant in primary cells versus cell lines.
thymidine analog mutations (TAMs) showed that the replication
of each of these variants was less efficient in primary human
macrophages than in T-cell lines (129). It is interesting that a
mutant virus with the four TAMs D67N, K70R, T215Y, and
K219Q actually had a replication advantage compared to wild-
type virus in unstimulated PBMCs; this difference was not ob-
served when the PBMCs were stimulated before infection (26).
Similar to the M184V mutant, the differences in replication fit-
ness observed under these conditions were associated with differ-
ences in processivity under limiting nucleotide concentrations.
Thus, it seems likely that differences in the replication fitness of
nucleoside resistance mutants are more likely to be observed in
cell types in which nucleotide pools are limited.
In contrast, no such impact of cell type was observed when
the replication fitness of HIV-1 mutants that are resistant to
556DYKES AND DEMETERCLIN. MICROBIOL. REV.
nonnucleoside reverse transcriptase inhibitors (NNRTIs) was
studied (4, 69, 94, 174). These findings are consistent with the
fact that these mutants do not appear to affect the processivity
of polymerization or nucleotide affinity (51, 69, 174). We are
not aware of published studies comparing the replication fit-
ness of mutants that are resistant to protease inhibitors in
different cell types. However, based on the known mechanisms
for drug resistance of this class of mutant, one would not
expect to see the pronounced effects of cell type on replication
fitness that are seen with nucleoside-resistant mutants.
in culture for extended periods of time, most assays of repli-
cation fitness utilize T-cell lines rather than primary cells.
Thus, these assays may overestimate the replication efficiencies
of isolates containing nucleoside resistance mutations.
Other differences among fitness assays. In addition to these
major differences in assay design, methods to determine the
amount of virus used to infect a culture-based fitness assay may
differ. The method used to determine virus inoculum can have
a significant impact on the apparent fitness of a strain in par-
allel infections, single-cycle assays, or growth competition as-
says if the relative proportions of test and reference variants at
a single time point after infection are compared to the propor-
tions in the original inoculum. One study rigorously evaluated
the correlation between different measures of virus input using
a number of different clinical isolates and found that the re-
verse transcriptase activity of the virus stock correlated better
with infectious titer than p24 antigen or quantitative viral RNA
assays (115). It is not clear whether this analysis included
strains containing drug resistance mutations in reverse trans-
criptase, which might alter the relationship between reverse
transcriptase activity and virus infectivity, so these findings may
not necessarily apply to drug-resistant strains of HIV-1.
Examples of specific assays. The only commercially avail-
able fitness assay is a parallel-infection, recombinant-virus, sin-
gle-cycle RC assay developed by Monogram Biosciences, Inc.
(formerly ViroLogic) (15). This assay, which is a variation on
Monogram’s phenotypic drug susceptibility assay (133), com-
pares test and reference strains in the absence of drug (Fig. 2).
An amplicon spanning the p7-p1-p6 cleavage sites in Gag, the
entire protease, and part of the reverse transcriptase (codons 1
to 313) is obtained from patient plasma and cloned in bulk into
an HIV-1 vector derived from the pNL4-3 infectious molecular
clone containing a luciferase reporter gene that replaces part
of env (133). Luciferase is expressed upon establishment of
infection and is a measure of viral infection (133). MLV en-
velope is supplied in trans during the production of the virus
stock, yielding pseudotyped virus. Since the RC assay is a
single-cycle assay and since luciferase detection can be auto-
mated, it has a high throughput. Luciferase quantitation also
has a wide linear range. The potential disadvantages of this
assay are that it measures the fitness contributions of the 3? end
of gag, protease, and part of reverse transcriptase only and uses
parallel infections, which may be less sensitive and more sus-
ceptible to variations in virus input than growth competition
assays. The use of a murine retroviral envelope instead of
HIV-1 could also possibly affect the relative fitness of mutants
that are resistant to protease and/or reverse transcriptase in-
hibitors, although this seems unlikely. The Monogram RC
assay has been the most widely used fitness assay in clinical
cohorts, and results correlating RC with clinical outcome will
be summarized later in this review.
There are several other types of assays developed by indi-
vidual research laboratories that have been used to quantify
the relative fitness of site-directed mutants or clinical isolates.
Here, we will briefly describe several different assays to illus-
trate the types of approaches that have been taken, recognizing
that this is not an exhaustive list.
An assay used by several laboratories is a multiple-cycle
recombinant-virus growth competition assay in which the rel-
ative proportion of test and reference strains is measured using
either bulk or clonal sequence analysis (4, 65, 69, 78, 86, 117,
119, 154, 155, 174). This type of assay is labor-intensive and
difficult to apply to large numbers of clinical samples but has
been commonly used to characterize the relative fitness of
site-directed drug-resistant mutants of HIV-1 in protease and
reverse transcriptase. Depending on the laboratory and the
specific mutants studied, either T-lymphocyte lines or primary
human PBMCs have been used as target cells. A recombinant-
virus growth competition assay in which test and reference
strains are quantified using real-time PCR of the hisD and
PLAP reporter genes has been used to study the relative fit-
nesses of mutants that are resistant to the nucleoside analog
zidovudine (81, 105) and the fusion inhibitor enfuvirtide (104).
A potential advantage of this assay is the greater dynamic
range and reduced labor of real-time PCR.
A whole-virus, multiple-cycle growth competition assay in
primary human PBMCs in which the relative proportion of test
and reference viruses is measured using an HTA has been used
to study the effects of env on fitness and disease progression
and to evaluate the relative fitnesses of different subtypes of
HIV-1 (6, 11, 140, 164). Because this growth competition assay
utilizes an intact viral isolate and primary human PBMCs, it
likely provides the closest approximation to HIV-1 replication
fitness in patients compared to other currently available assays,
but its labor-intensive nature limits its use for large-scale stud-
ies correlating fitness with clinical outcomes.
Some laboratories have utilized multiple-cycle, whole-virus,
parallel infections to measure growth kinetics in primary hu-
man PBMCs (27, 163). This type of assay offers the advantages
of using primary cells and an intact virus isolate as well as
improved throughput compared to growth competition assays.
If replication fitness is estimated using the slope of virus ex-
pansion after initial infection, this will markedly reduce the
influence of virus inoculum on apparent replication fitness.
However, a theoretical concern is assay-to-assay variation,
since there is no internal control as in a growth competition
assay. These assays have demonstrated associations of replica-
tion fitness with viral load and will be discussed in more detail
later in this review.
Another recently reported assay developed by Tibotec and
Virco is a high-throughput assay to measure replication rates
in parallel infections. Eight serial dilutions of virus are made,
with six time points tested per sample, and the measurements
are normalized for viral input by multiplying by the dilution.
Viral replication is measured using a cell line containing an
enhanced GFP (EGFP) reporter gene under the control of an
HIV long terminal repeat (LTR). Infection with HIV results in
the activation of LTR and EGFP expression. The log10of the
VOL. 20, 2007 CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS557
product of fluorescence and viral dilution is linear with respect
to time, and the slope of this line is the replication rate, which
is independent of virus inoculum (146a). The very high
throughput of this assay would make it very amenable to use in
a clinical setting, although this use of the assay has not yet been
Some recent publications have described fitness assays that
utilize flow cytometry as a method to determine virus growth
and variant proportions. The potential advantages of such as-
says are higher throughput and the ability to monitor the
spread of infection at a cellular level. Zhang and coworkers
designed a recombinant-virus single-cycle assay in which the
GFP gene replaces env in the infectious HIV-1 molecular clone
pNL4-3; test and reference strains were then compared in
parallel infections (184). The use of red fluorescent proteins
(DsRed2) and EGFPs as markers in a multiple-cycle recom-
binant-virus growth competition assay in primary human
PBMCs has also been described and was used to characterize
the relative fitness of mutants that are resistant to the fusion
inhibitor enfuvirtide (125).
A flow cytometry-based recombinant-virus multiple-cycle
growth competition assay has also been developed in which the
test and reference viral vectors contain the mouse Thy1.1 or
Thy1.2 allele in place of nef. The Thy gene products are ex-
pressed on the surface of infected cells early during viral in-
fection and can be detected by commercially available fluores-
cence-labeled monoclonal antibodies (57). There was an
excellent correlation of relative fitness of different drug-resis-
tant mutants, as measured by flow cytometry, compared to
direct and clonal sequence analysis, indicating that the linkage
between the resistance mutations and the reporter gene was
not disrupted by recombination (57). Such dual-marker flow
cytometry-based assays are exciting in that they have the po-
tential to allow the use of growth competition assays to mea-
sure replication fitness in larger-scale clinical trials.
Other Assays To Measure Fitness
An interesting modification to the cell culture growth
competition assays described above is the rapid cell turnover
assay (172). In contrast to standard cell culture assays in
which half the cultured cells are typically replaced every 4 to
7 days, 90% of the cultured cells are replaced with fresh
uninfected cells every 2 days, a time interval similar to the
life span of T cells in patients. Under these conditions, it has
been shown that the fitness of the highly pathogenic simian
immunodeficiency virus clone Mne170 was higher than that
of its parental clone CL8, whereas under normal culture
conditions, they were indistinguishable. The result obtained
under rapid cell turnover conditions is more consistent with
the relative pathogenicity of the two clones in animals, sug-
gesting that rapid cell turnover conditions may be a better
predictor of viral pathogenicity in clinical infection. This
interesting finding needs to be confirmed with more exten-
sive studies of HIV-1 isolates.
Although more labor-intensive and much less commonly
used, HIV-1 replication fitness has also been measured in
SCID-hu mice that are reconstituted with human peripheral
blood leukocytes and then infected with patient isolates. This
methodology has been used to look at the properties of isolates
with resistance mutations in protease and reverse transcriptase
(91a, 135, 159). An interesting feature of this assay is that
CD4?T-cell depletion can be evaluated, suggesting that this
assay may be able to evaluate some aspects of virus pathoge-
nicity that are not captured in traditional cell culture-based
assays of replication fitness. The disadvantages are that it is
much more labor-intensive and costly than cell culture assays,
and therefore, the ability to correlate viral replication fitness
with clinical outcomes in significant numbers of clinical sam-
ples is extremely limited. Nonetheless, this assay has the po-
tential to be a very interesting research tool to better under-
stand the relationship between HIV-1 replication efficiency
Measurements of viral fitness have also been made by assaying
the prevalence of TAMs in viral quasispecies present in plasma
from patients not receiving antiretroviral therapy (71, 72). Allele-
specific PCR assays or clonal sequence analysis was used to quan-
tify the relative proportions of the different drug-resistant variants
over time, and mathematical modeling was used to determine the
fitness gain of the revertant virus over the mutant. An important
advantage of this approach is that it takes into account selective
pressures present in patients. However, this approach assumes
that the specific mutation being assayed has the same effect in
each viral genome and does not take into account the potential
modulating effects of other mutations on viral fitness. For exam-
ple, if the prevalence of T215Y in a patient did not decrease after
the discontinuation of the drug due to a selective advantage con-
ferred by a nonresistance mutation at another codon, this fitness
value could erroneously be attributed to T215Y. In addition, the
replication properties of the “wild-type” reference strain that
overgrows the mutant will differ from patient to patient. There-
fore, one must use caution in attributing fitness values obtained
from such studies solely to the resistance mutation(s) being as-
MATHEMATICAL APPROACHES TO QUANTIFYING
Although cell culture-based fitness assays can provide
qualitative information regarding the replication fitness of
one variant relative to that of another, determining whether
such in vitro measures of fitness correlate with clinical out-
come requires reliable methods to quantitate the degree to
which fitness is altered relative to the reference strain. Re-
liable quantitation will also be required if fitness assays are
to be used in clinical practice. Unfortunately, there has been
no clear consensus on how to quantify relative fitness, and
publications differ in their mathematical definitions of a
fitness coefficient. This lack of consensus has led to confu-
sion in the literature and has further limited the ability to
compare results from different studies. We will discuss
methods to quantify fitness for multiple-cycle and single-
cycle assays separately, since investigators have taken dif-
ferent approaches for these two types of assays.
According to population genetics, the relative fitness of a
variant, defined as 1 ? s, is the relative contribution of that
variant to the next generation; s is defined as the selection
558DYKES AND DEMETERCLIN. MICROBIOL. REV.
coefficient. If a mutant is more fit than the reference strain,
1 ? s will be greater than 1, and successive generations will
have an increasing proportion of progeny derived from that
mutant. If a mutant is less fit than the reference strain, 1 ? s
will be less than 1, and the prevalence of that mutant will
decline over time relative to the reference strain.
A confusing aspect of the literature on fitness is that the
value of 1 ? s has sometimes been defined differently (re-
viewed in more detail in reference 181). Using viral dynamic
models, Wu and coworkers attempted to clarify the different
definitions of fitness, proposing standard nomenclature that
distinguishes different approaches to measuring fitness and
providing approaches to perform statistical evaluations (181).
In these viral dynamic models, it is assumed that only two
variants are present (i.e., no recombinants) and that there is a
constant number of target cells over time (i.e., a large excess of
susceptible target cells). Three parameters were defined, each
of which takes a different approach to quantifying fitness: “log
relative fitness,” “log fitness ratio,” and “production rate ra-
tio.” The log relative fitness value, d, is equal to the natural log
of 1 ? s, where s is the selection coefficient defined by popu-
lation genetics theory (Table 2). d is equivalent to the differ-
ence between the net growth rates of the mutant and reference
strains (Table 2). In contrast, the log fitness ratio, r, is the ratio
of the net growth rates of the mutant and reference strains.
The production rate ratio, p, differs from the two previous
parameters in that it is the ratio of the production rates of the
mutant and reference strains and assumes that the life span of
infected cells does not contribute to fitness. p is equivalent to
1 plus the selection coefficient defined by Maree and coworkers
(112), which differs from the selection coefficient defined by
population genetics theory.
An important point is that d and r compare the growth rates
of the mutant and the wild type at the same time point and
therefore do not need to be corrected if the culture is diluted
during the course of the growth competition assay. In addition,
p must incorporate the total expansion of the two viral variants;
use of relative proportions of variants without accounting for the
degree of viral expansion will give incorrect values. A publicly
available website provides calculators and guidance on the use
and statistical analysis of these different fitness parameters (http:
The Monogram Biosciences assay expresses RC of a test strain
as a percentage of the wild-type reference virus (14, 15, 41). All
values are normalized for the efficiency of the transfections used
to produce the virus stocks. For example, if infection with a
recombinant virus derived from a clinical sample leads to half of
the luciferase activity of the wild-type reference strain, and if the
test and reference virus stocks had equal transfection efficiencies
and were used in equal volumes in the infection, the RC value of
the clinical isolate would be reported as 50%. There are no pub-
lished data on how this method of normalizing virus inoculum
compares to other methods.
EFFECTS OF SPECIFIC DRUG RESISTANCE
MUTATIONS ON FITNESS
A major focus of early literature on HIV-1 replication fitness
was an evaluation of the impact of specific drug resistance
mutations on viral fitness. The majority of these studies utilized
site-directed mutants of laboratory strains, although some
studies also evaluated clinical isolates (either whole virus or
recombinant viruses containing a genome segment of the clin-
ical isolate). This continues to be an important area of study as
new drugs and combination therapies are introduced into clin-
ical practice. In general, nearly all the drug resistance muta-
tions studied adversely impact HIV-1 replication fitness to
some extent, although different mutations vary in the magni-
tudes of their effect. Since replication fitness is proposed to
influence the prevalence of an HIV-1 variant in patients and
may also impact clinical outcome, an understanding of the
relative impact of different drug-resistant mutations may pro-
vide important, clinically relevant information. The following
section will describe what is known about the replication fitness
of specific drug resistance HIV-1 variants conferring resistance
to drugs that are currently FDA approved, organized accord-
ing to the drug class that is affected. Because of the variations
in assay design and approaches to calculating fitness values
summarized above, much of this discussion will be qualitative,
with comparisons among mutants limited primarily to those
that were studied with the same methods (see Table 3 for a
summary of the effects of the single drug resistance mutations
discussed below on fitness).
Mutations Conferring Resistance to Reverse
nRTIs. Nucleoside and nucleotide reverse transcriptase inhib-
itors (nRTIs) are competitive inhibitors of nucleotides that are
normally incorporated during the synthesis of the viral genome.
nRTIs bind to the polymerase active site of reverse transcriptase
and competitively inhibit the synthesis of proviral DNA by acting
as chain terminators during synthesis (for a review of this class of
drugs, see reference 171). Several nRTI resistance mutations can
confer cross-resistance to more than one nRTI.
TABLE 2. Different approaches to calculating fitness in
p ? km/kw
ln?Tm(t2)/Tm(t1)? ? ?m?t ? ln2
ln?Tw(t2)/Tw(t1)? ? ?w?t ? ln2
ln?Tm(t2)/Tm(t1)? ? ln2
ln?Tw(t2)/Tw(t1)? ? ln2
r ? gm/gw
d ? gm? gw
1 ? s ? exp(d)
aNomenclature proposed by Wu et al. (181). Computational tools are avail-
able at the following website: http://www.urmc.rochester.edu/bstools/vfitness
bDefinition of terms used in these formulas is as follows: Tmis the number of
cells infected by infectious mutant virus, Twis the number of cells infected by
infectious wild-type virus, ?mand ?ware death rates of infected mutant and
wild-type cells, respectively, kmand kware infection rates of mutant and wild-
type virus, respectively, gmand gware net growth rates of mutant- and wild-type-
infected cells, respectively, and s is the selection coefficient, as defined by pop-
ulation genetics theory.
VOL. 20, 2007 CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS 559
(i) M184V. The replication fitness deficit conferred by the
M184V mutation in reverse transcriptase, which causes high-
level resistance to the cytosine analogs lamivudine and emtric-
itabine, has been the subject of extensive study (reviewed in
reference 132). M184V occurs frequently in viral isolates from
patients failing lamivudine or emtricitabine therapy (63, 153).
M184V has a fitness similar to that of the wild type in most
T-cell lines where nucleotide concentrations are high (10) but
reduced fitness in primary cells that have limited nucleotide
pools, such as PBMCs and macrophages (3, 10, 129). Of note
is that there are some more-recent studies that have found
reduced fitness of this mutant in cell lines (35, 48).
A multitude of biochemical abnormalities have been proposed
to explain the reduced fitness conferred by the M184V mutation,
including decreased processivity of polymerization that is aggra-
vated by low nucleotide concentrations, decreased initiation of
TABLE 3. Effects of drug resistance mutations on HIV-1 replication fitness
Fitness assay used (cell type)a
Fitness relative to wild
K65RMultiple cycle, parallel cultures (cell line)
Single cycle, parallel cultures (cell line, primary cells ?PBMCs?)
Multiple cycle, growth competitionb(cell line)
Single cycle, parallel cultures (primary cells ?macrophages?)
Multiple cycle, growth competition (primary cells ?PBMCs?)c
Single cycle, parallel cultures (cell line)d
Multiple cycle, parallel cultures (cell line)
Multiple cycle, parallel cultures (primary cells [PBMCs])
Multiple cycle, growth competitionb(primary cells [PBMCs])
Multiple cycle, growth competitionb(cell line)
Multiple cycle, parallel cultures (cell line)
Single cycle, parallel cultures (cell line, primary cells ?PBMCs?)
Multiple cycle, parallel cultures (primary cells ?PBMCs?)
Single cycle, parallel cultures (primary cells ?macrophages?)
Single cycle, parallel culture (cell line)d
Multiple cycle, growth competitionb(cell line)
Multiple cycle, growth competitionb(cell line)
Multiple cycle, growth competitionb(cell line)
Multiple cycle, parallel infections (cell line)
Multiple cycle, parallel infections (cell lines, primary cells ?PBMCs?)
Multiple cycle, growth competitionb(cell line)
Slight reduction (48)
Slight reduction (106)
K103N Multiple cycle, growth competitionb,e(cell line)Similar (33, 57, 94)
Y181C Multiple cycle, growth competitionf(cell line)
Multiple cycle, growth competitionb,g(cell line)
Multiple cycle, growth competitionb,g(cell line)
Single cycle, parallel infectionsd(cell line)
Multiple cycle, growth competitionb,e; single cycle, parallel
infections (cell line)
Multiple cycle, growth competitionb,e; single cycle, parallel infections
SCID-hu mouse model
Single cycle, parallel infections
SCID-hu mouse modelh
Multiple cycle, growth competitioni(cell line, primary cells
Multiple cycle, growth competitioni(cell line, primary cells
Multiple cycle, growth competitioni(cell line, primary cells
Reduced (4, 33)
Reduced (57, 82, 174)
Similar (57, 119, 130)
Reduced (57, 119, 130)
V38M or AReduced (104)
aAll assays tested site-directed mutants of laboratory strains of HIV-1, unless stated otherwise.
bRelative proportions of the mutant and wild type were measured by sequence analysis.
cIntact clinical isolates were tested. Relative proportions of the mutant and wild type were measured by real-time PCR of env.
dMonogram Biosciences RC assay using patient-derived recombinant viruses.
eRelative proportions of the mutant and wild type were measured using flow cytometry to detect Thy1.1 and Thy1.2 reporter genes.
fRelative proportions of wild-type and mutant strains, which were selected for in the presence of nevirapine, were determined by direct sequence analysis. Relative
proportions of the wild type and a site-directed mutant of Y181C were determined by HTA.
gRelative proportions of the mutant and wild type were measured using an allele-specific real-time PCR assay.
hClinical isolates from patients whose virus had acquired the V82A mutation were studied.
iRelative proportions of the mutant and wild type were measured using real-time PCR to detect hisD and PLAP reporter genes.
560DYKES AND DEMETERCLIN. MICROBIOL. REV.
minus-strand DNA synthesis from tRNA3
strand DNA synthesis from the HIV RNA polypurine tract se-
quence, reduced binding affinity for nucleotides, improved fidelity
of nucleotide incorporation, and slowed rates of RNase H cleav-
age (10, 48, 49, 173, 181a).
There has been a great deal of focus in the literature on the
reduced replication fitness of M184V and its proposed clinical
implications. Early observations of viral load rebound during
lamivudine monotherapy and lamivudine-containing regimens
demonstrated that plasma HIV-1 RNA concentrations re-
mained lower than pretherapy levels despite the uniform pres-
ence of M184V in plasma viruses (132, 153). However, other
studies have shown that lamivudine does exert some antiret-
roviral effect on the M184V mutant, suggesting that the incom-
plete viral rebounds that occur may represent residual antiviral
activity rather than selection for a mutant with reduced fitness
(139). Studies of selective lamivudine treatment interruptions
demonstrate increases in viral load before reversion of the
M184V mutant, also supporting the hypothesis that lamivudine
retains antiretroviral activity against M184V (28).
In summary, the M184V mutant is clearly reduced in repli-
cation fitness, but these reductions appear to be no greater
than those of many other drug-resistant mutants when mea-
sured in cell culture assays. Although the M184V mutant might
be attenuated in ways that are not detected by standard repli-
cation fitness assays, many of the clinical observations support-
ing this hypothesis can also be explained by the persistent
antiviral activity of lamivudine. It thus seems less likely that
M184V has unique properties that make it substantially less
pathogenic than other drug-resistant mutants.
(ii) TAMs. Using growth competition assays in cell lines with
site-directed mutants, the TAMs T215Y and M41L and the
M41L/T215Y double mutant were all less fit than the wild type
(78). K70R, which is usually the first TAM to emerge during
zidovudine therapy, was substantially more fit in this assay than
T215Y (78). This finding suggests that the reduced fitness of
T215Y may explain the delay in this mutant’s emergence
relative to K70R, although the fact that T215Y requires a
two-nucleotide change also likely contributes to its delayed
A quadruple TAM mutant (D67N/K70R/T215Y/K219Q) is
unusual in that it has been shown to have a replication advan-
tage over the wild type in PBMCs that were stimulated before
infection but not if they were stimulated after infection, sug-
gesting that this improved fitness relative to wild-type virus was
dependent on low-nucleotide pools (26). This observation was
supported by biochemical studies that demonstrated that the
quadruple TAM mutant had increased processivity of polymer-
ization relative to that of the wild type in the presence of low
nucleotide concentrations (26).
Observations of patients failing zidovudine-containing regi-
mens have identified two preferred combinations, or pathways,
of TAMs: M41L/L210W/T215Y (TAM-1) and D67N/K70R/
T215F/K219Q (TAM-2) (77, 111). The TAM-1 pathway is
more common than TAM-2 in clinical isolates and differs from
TAM-2 in that it causes cross-resistance to tenofovir. Using a
recombinant-virus growth competition assay in a cell line,
T215F was less fit than T215Y, which may explain why T215F
rarely occurs by itself and is less common than the T215Y
mutation (81). L210W reduced the fitness of D67N/K70R/
Lys, decreased plus-
K219Q in the presence or absence of drug but increased the
fitness of M41L/T215Y in the presence of drug, which may
explain why this mutation is seen only as part of the TAM-1
Studies of patients have demonstrated that the T215Y mu-
tant can revert to intermediate mutations, such as T215S,
T215D, or T215C, in the absence of drug. A study of a patient
newly infected with a T215Y-containing strain of HIV-1 dem-
onstrated the gradual replacement of T215Y by T215S (72),
indicating that the revertant mutant was more fit than T215Y
in this patient. Using viral dynamic modeling in which the ratio
of their production rates was measured, the fitness of T215S
was estimated to be 0.4 to 2.3% better than that of T215Y (72).
In another individual recently infected with a drug-resistant
isolate containing M41L and a mixture of T215Y/T215D/
T215S, T215D and T215S were estimated to be 10 to 25% and
1% more fit, respectively, than T215Y (71). The improved
fitness of the T215D/T215S mutants relative to T215Y was also
observed in another study of site-directed mutants (66).
(iii) L74V. L74V, which confers resistance to both di-
danosine and abacavir, has reduced fitness in studies of site-
directed mutants relative to wild-type virus and K70R using
growth competition assays in primary human PBMCs and a cell
line (35, 154). It has been shown that L74V in the context of
the NL4-3 laboratory strain has an 11% loss of fitness com-
pared to that of the wild type or K70R (154). Biochemical
studies have shown that this replication decrease is associated
with a reduction in the incorporation of nucleotides compared
to the wild type and a decrease in the initiation of minus-strand
and plus-strand DNA synthesis from the tRNA3
rine tract primers (47, 49). A reduced ability of L74V viruses to
synthesize proviral DNA was demonstrated in cell culture (61).
(iv) K65R. K65R, which confers resistance to the nucleotide
analog tenofovir, has been shown to have a reduction in fitness
when introduced into laboratory strains, as well as in the con-
text of patient sequences, as measured by the Monogram Bio-
sciences RC assay (35, 46, 179). Similar findings were also
obtained using whole isolates obtained from patients failing
tenofovir therapy (175). One study using single-cycle parallel
cultures found a decreased fitness of K65R in primary macro-
phages but not in a cell line or primary human PBMCs (129).
The replication defect of K65R correlates with a decrease in
processivity of polymerization and in the polymerization rate,
kpol, of purified reverse transcriptase (46, 179).
(v) Q151M complex. The Q151M complex (A62V, V75I,
F77L, F116Y, and Q151M) confers resistance to all FDA-
approved nRTIs except tenofovir (87) and yet develops much
less frequently than T215Y (reviewed in reference 171). Both
the single Q151M mutant and the full Q151M complex were
first shown in parallel infections (in cell lines and PBMCs) to
have a replication fitness similar to that of the wild type (106),
but later competition experiments showed that the full Q151M
complex was more fit than the Q151M single mutant and that
both were actually more fit than the wild type, which is incon-
sistent with their low frequency in patients (93). Of note is that
the mutation Q151M, like T215Y, requires a two-nucleotide
change from the wild-type sequence. It is interesting that the
replication fitness of 151L and 151K, the intermediate muta-
tions leading to 151M, are much lower than that of the wild
type, suggesting that the genetic barrier to developing Q151M
VOL. 20, 2007 CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS 561
is due in part to the low fitness of the intermediate mutants
NNRTIs. NNRTIs are a structurally diverse group of com-
pounds that bind to a hydrophobic pocket adjacent to the
polymerase active site of reverse transcriptase. They inhibit
reverse transcriptase by causing an allosteric change in the
enzyme that interferes with polymerization. There are cur-
rently three FDA-approved NNRTIs: nevirapine, delavirdine,
and efavirenz (for a review of NNRTIs, see reference 89).
Efavirenz is widely used as part of first-line combination reg-
imens for treatment-naive patients, and nevirapine is fre-
quently used for the prevention of mother-to-child transmis-
sion during pregnancy (44, 45).
These drugs are potent inhibitors of HIV-1 replication, but
NNRTI-resistant variants of HIV-1 develop rapidly if virologic
suppression is not complete. The most commonly seen resis-
tance mutations in clinical isolates from patients failing nevi-
rapine and delavirdine therapy are K103N and Y181C; K103N
is most common during efavirenz failure (9, 43, 145). However,
several mutations, such as G190S, P236L, and V106A, are
equally resistant to one or more of these drugs but occur
uncommonly in clinical isolates. K103N is postulated to de-
velop commonly in clinical isolates because it has minimally
reduced replication fitness in the absence of drug (33, 57, 69,
94). Although one group of investigators has reported that
Y181C has a slight fitness advantage over the wild type (84),
two other groups have found that Y181C is modestly less fit
than K103N and the wild type (4, 33). In contrast to K103N
and Y181C, uncommonly occurring NNRTI resistance muta-
tions confer significantly greater replication deficits (4, 33, 69,
94, 174). Some studies in which fitness assays were performed
in the presence of efavirenz have shown that these replication
defects relative to K103N persist in the presence of drug (69,
Less-fit NNRTI-resistant mutants have greater reductions in
rates of RNase H cleavage, suggesting that this biochemical
abnormality contributes to their reduced replication fitness (4,
5, 69, 174). In addition, recent studies suggest that reduced
initiation from the tRNA3
duced replication efficiency of at least some NNRTI-resistant
mutants (174; for a more detailed review of the biochemical
effects of NNRTI resistance mutations on reverse transcriptase
function, see reference 52). When NNRTI-resistant mutants
have been compared directly, the magnitude of their fitness
deficit generally correlates with the magnitude of their bio-
chemical effects on reverse transcriptase function.
Studies evaluating how long NNRTI resistance mutations
persist after discontinuation of an NNRTI have also been used
to estimate relative fitness. For example, NNRTI resistance
mutations such as K103N and Y181C, which have minimal
reductions in fitness, can persist for up to a year after the
withdrawal of therapy (88).
More than one NNRTI resistance mutation may occur, par-
ticularly with more prolonged virologic failure, raising the
question of how these mutations interact to affect HIV-1 rep-
lication fitness. One study of nevirapine-resistant mutants
demonstrated that triple mutants were generally less fit than
double mutants, which were less fit than single mutants (33).
However, the fitness values of specific single and double mu-
tants did not always predict the relative fitness of the corre-
Lysprimer also contributes to the re-
sponding triple mutant, suggesting that the relative fitness of
NNRTI-resistant mutant combinations is not merely a function
of their number. In addition, some quadruple mutants were
more fit than the triple and some of the double mutants (33).
The fact that the number of NNRTI resistance mutations does
not always predict replication fitness is also supported by stud-
ies of secondary mutations conferring resistance to efavirenz
that develop after the emergence of K103N (94). Those studies
indicate that which specific secondary mutation is present has
a significant impact on the relative fitness of the double mutant
and that the relative fitness of the double mutants generally
correlates with their frequency in clinical samples (94).
Thus, there is evidence from studies of both nRTI and
NNRTI resistance mutations that relative replication fitness, as
measured in a cell culture assay in the absence of drug, gen-
erally correlates with mutant prevalence during virologic fail-
ure in patients. This finding is compatible with predictions
based on theoretical models of viral dynamics (31). A predic-
tion of this model is that HIV-1 replication fitness in the
absence of drug influences the relative frequency of randomly
generated mutants before therapy initiation and, thus, the like-
lihood that they will emerge during virologic failure (31). Stud-
ies with some poorly fit NNRTI-resistant mutants demonstrate
that the selective disadvantage relative to K103N persists even
in the presence of drug. These studies provide support for the
concept that relative fitness correlates with mutant prevalence
in patients and suggest that this information may be helpful
during preclinical drug development, particularly in the search
for newer-generation NNRTIs that do not select for K103N or
Mutations Conferring Resistance to Protease Inhibitors
Protease inhibitors act as competitive inhibitors of protease
cleavage, which is required for the posttranslational processing
of the gag and pol gene products (reviewed in reference 85).
They are widely used in combination with nRTIs as initial
therapy for treatment-naive patients and as part of combina-
tion regimens for treatment-experienced patients (reviewed in
reference 20). High-level resistance to protease inhibitors usu-
ally requires several mutations in the protease gene. Protease
resistance mutations are divided into two groups: major and
minor mutations (87). It should be noted at the outset that the
classification into major and minor mutations is not always
clear-cut, and some mutations have been placed into both
categories, depending on the specific protease inhibitor. None-
theless, this classification scheme has proven useful for many of
the protease inhibitor resistance mutations.
Major mutations (also referred to as primary mutations) are
generally those that are acquired early in failure or which
directly affect drug binding or drug susceptibility. Examples of
major mutations are D30N, I50V/L, V82A/F/T/S, I84V, and
L90M. Some of these major mutations confer resistance pri-
marily to specific protease inhibitors, such as D30N for nelfi-
navir and I50L for atazanavir. These major mutations are gen-
erally located in the active site of the HIV-1 protease.
Minor mutations (also referred to as secondary mutations)
usually develop after the emergence of major mutations and by
themselves do not increase the level of drug resistance (87).
Examples of minor mutations are L10I/R/V, A71V/T, and
562 DYKES AND DEMETERCLIN. MICROBIOL. REV.
V77I. Minor mutations are generally located away from the
active site of protease. Some minor mutations may be present
in treatment-naive patients, although usually fewer than three
minor resistance mutations are observed in this patient popu-
lation (42, 95).
Major protease inhibitor resistance mutations. The devel-
opment of HIV-1 resistance to the protease inhibitor nelfinavir
occurs by the acquisition of the primary resistance mutation
D30N or L90M (128). D30N is more resistant to nelfinavir
than L90M but is susceptible to other protease inhibitors,
unlike L90M. The fitness of D30N is quite impaired and is
substantially lower than that of either the wild type or L90M;
L90M is only modestly impaired in replication (57, 119, 130).
Thus, the higher degree of nelfinavir resistance appears to
drive the preferential selection for D30N, whereas the rela-
tively preserved fitness of L90M appears to favor its emergence
despite its lower level of drug resistance. D30N was also found
to be significantly impaired in its ability to infect SCID-hu mice
transplanted with human thymus (91a). The D30N mutant
protease has reduced catalytic efficiency, which presumably
explains its decreased fitness (30). In another study, D30N
virus demonstrated processing of Gag and Gag-Pol proteins
that was substantially reduced compared to that of L90M,
consistent with their relative replication fitness (161). D30N
and L90M rarely occur together in clinical isolates, most likely
due to the fact that the double mutant is profoundly reduced in
replication fitness (130, 161).
G48V, which is a major mutation conferring resistance to
saquinavir, was shown to have decreased fitness in a single-
cycle assay (109). I50L, which confers resistance to atazanavir,
also has reduced fitness in parallel infections (34). The I50V
mutation, which is associated with resistance to amprenavir,
lopinavir, and darunavir, showed a marked reduction in fitness
in a single-cycle assay; I84V, which is more broadly cross-
resistant, showed a similar reduction in fitness in the same
assay (138). Another group did not identify a clear reduction in
the replication of the I84V mutant but did demonstrate im-
paired proteolytic processing by this mutant (36). V82T, which
is one of the substitutions at codon 82 that confer broad cross-
resistance, was also significantly reduced in fitness compared to
that of the wild-type virus (118). V82A, which confers low-level
but broad cross-resistance, did not demonstrate significantly
reduced fitness in a single-cycle assay but did have a replicative
advantage compared to wild-type virus in the presence of drug
(109). Consistent with these results from site-directed mutants
are studies of serial clinical isolates obtained from patients
failing protease inhibitor therapy that have correlated the de-
velopment of reduced fitness in the Monogram RC assay with
the emergence of V82A, I84V, or L90M (14). In addition,
clinical isolates containing V82A had reduced replication in
the SCID-hu mouse model (135).
In summary, most major protease inhibitor resistance mu-
tations confer some replication defect, although in some
cases, this defect is modest. For those mutants that have
been studied, these replication deficits appear to be associ-
ated with reduced catalytic efficiency, consistent with their
location at or near the active site of protease. The greatest
deficits appear to be due to those mutations, such as D30N
and G48V, which confer higher-level resistance that is re-
stricted to one or a few protease inhibitors. Other primary
mutations that confer lower-level, broader cross-resistance
appear to result in smaller replication deficits, although
these two types of mutants have not been directly compared
in a single study.
Minor protease inhibitor resistance mutations. In contrast
to major mutations, minor protease inhibitor resistance muta-
tions by themselves generally have little or no adverse effect on
HIV-1 replication fitness. For example, little or no reductions
in fitness were seen for L63P, M46I (118), and L10F (138). In
addition, at least some minor resistance mutations can com-
pletely or partially improve the replication deficits conferred
by major resistance mutations. Some of these interactions can
be quite specific; for example, L63P can compensate for the
reduced fitness of L90M but not D30N (119). A71V can also
compensate for the reduced fitness of D30N (130).
It should be noted, however, that some combinations of
major and minor resistance mutations actually decrease fitness,
also suggesting that these mutational interactions are specific
(138). A recently reported study in which a large number of
major and minor mutations (31 single and 42 double mutants)
were evaluated for infectivity and fitness with the Monogram
RC assay indicates that most minor mutations can compensate
for the fitness deficit conferred by most major mutations. This
finding suggests that the mechanism(s) for compensation are
probably nonspecific for double mutants; more complicated
combinations of mutations were not studied (78a).
Consistent with most of these studies of site-directed mu-
tants are studies of clinical isolates obtained from patients
failing protease inhibitor therapy that demonstrated an initial
loss of fitness and increase in resistance associated with the
emergence of major mutations, followed by progressive in-
creases in fitness with the accumulation of minor mutations
(14, 126). However, the improvements in fitness were not al-
ways complete, suggesting that HIV-1 protease may be limited
in its ability to become both highly resistant and highly fit (14).
It has been shown that recombinant viruses containing pro-
tease genes from clinical isolates tend to evolve in cell culture
in the absence of drug only if the initial replication fitness, as
measured by the Monogram RC assay, is substantially reduced
(166). The paths to improved fitness involved either a reversion
of the major resistance mutation(s) or the acquisition of com-
pensatory mutations. Recombinant viruses that replicated sim-
ilarly to or better than the wild type did not evolve, suggesting
that there were no available evolutionary pathways to further
increase fitness (166).
For some combinations of protease inhibitor resistance mu-
tations present in clinical samples, the reversion of any one
mutation in the combination results in virus that is less fit than
virus with all the mutations intact, indicating that the virus
would have to go through a fitness trough in order to revert
(167). This finding could explain the persistence of what ap-
pear to be unfit mutants in patients after the discontinuation of
The fitness studies demonstrating compensation by minor
resistance mutations are also supported by biochemical analy-
ses. For example, one study demonstrated that M46I and L63P
by themselves augment the catalytic efficiency of HIV-1 pro-
tease and compensate for the reductions in catalytic efficiency
conferred by the major mutations V82A and I84V (152). When
both M36I and A71V are introduced into a protease contain-
VOL. 20, 2007CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS 563
ing D30N, the triple mutant demonstrated improved catalytic
efficiency compared to the single and double mutants as well as
wild-type protease (30).
Hypersusceptibility to protease inhibitors, defined as a 50%
inhibitory concentration of ?0.4-fold compared to the wild
type, can occur in treatment-experienced patients and some
treatment-naive patients and is associated with reduced viral
replication fitness (23, 120, 144, 185). The genotypes respon-
sible for the correlation of hypersusceptibility with replication
fitness are still not fully understood, and more studies are
needed to determine if the same mutations are responsible for
both hypersusceptibility and reduced fitness.
Mutations Conferring Resistance to Entry Inhibitors
Enfuvirtide (T-20) is the first FDA-approved drug belonging to
step in the viral life cycle when the virion attaches and fuses to the
cell membrane. Enfuvirtide is a 36-amino-acid peptide that mim-
ics the structure of the heptad repeat 2 (HR-2) domain of gp41;
enfuvirtide binds to the HR-1 domain of gp41, thus disrupting an
intramolecular interaction required for the fusion of the viral
envelope with the cell membrane (75). When HIV-1 is passaged
in the presence of enfuvirtide in cell culture, mutations at amino
acids 36 to 38 of HR-1 emerge, which confer resistance to enfu-
virtide (146). In patients failing enfuvirtide-containing regimens,
mutations at codons 36 to 45 and the double mutations N42T/
N43K and V38A/N42T emerge and have been shown to confer
cell culture (37T, 38 M, and 36S/38 M) are less fit than the wild
type in the absence of drug (104). The mutation combinations
selected in patients were also shown to be less fit than the wild
type, with a hierarchy as follows: wild type ? N42T ? V38A ?
N42T/N43K ? N42T/N43S ? V38A/N42D ? V38A/N42T ?
V38E/N42S. The reduced fitness conferred by enfuvirtide resis-
tance mutations in HR-1 is most likely due to their impairment in
study also found that some of these enfuvirtide-resistant mutants
conferred enhanced sensitivity to neutralizing antibodies, suggest-
ing another way in which the acquisition of enfuvirtide resistance
may confer a selective disadvantage to the virus.
HIV-1 isolates from patients who are enfuvirtide naive have a
range of susceptibilities to enfuvirtide. The reduced susceptibility
observed in some isolates from enfuvirtide-naive patients is not
thought to be clinically significant, since no reduction in the viro-
logic response to enfuvirtide in patients whose isolates demon-
strate this property could be demonstrated (121). Interestingly,
envelope sequences from enfuvirtide-naive patients that had re-
duced susceptibility to enfuvirtide but that did not have known
resistance mutations had preserved fitness, using a growth com-
petition assay in PBMCs, in contrast to resistant mutants that
develop during treatment failure (125).
MUTATIONAL INTERACTIONS THAT AFFECT FITNESS
Reverse transcriptase. (i) Interactions among nRTI resis-
tance mutations. We have already described some interactions
among nRTI resistance mutations that typically cluster to-
gether during the failure of a specific nRTI. For example,
L210W has different effects on fitness depending on with which
TAM pathway it is associated (81). A mutant with the TAMs
M41L and T215Y is more fit than M41L alone, offering an
explanation as to why M41L is rarely observed without T215Y
(78). The mutations that are part of the Q151M complex in-
teract to improve the fitness of the Q151M single mutant, also
providing an explanation for the preferred association of these
In addition, there are examples of interactions among nRTI
resistance mutations that confer resistance to different nucle-
oside analogs. L74V and K65R, which are rarely found to-
gether in clinical isolates, have markedly antagonistic effects on
viral fitness that correlate with reductions in the ability to
utilize normal nucleotides during DNA synthesis (47). Simi-
larly, K65R and M184V also have antagonistic effects on fit-
ness that are associated with abnormalities in nucleotide in-
corporation (46). The combination of K65R and the Q151M
complex reduced fitness compared with either mutant in iso-
lation and reduced the catalytic efficiency of reverse transcrip-
tase. In contrast, the Q151M complex did not reduce the fit-
ness of M184V, suggesting that the combinations of nRTIs
prescribed to the patient may influence whether the Q151M
complex develops (66a). A recent report has demonstrated
that the reduced fitness of M184V is improved when placed in
combination with the TAM-2 cluster of mutations; no such
effect is seen when M184V is combined with TAM-1 mutations
(35). The two clusters of TAM mutations had opposite effects
when combined with K70R (35).
A recent report, which documented the co-occurrence of
L74V, Q151M, and a deletion at codon 70 (?70) in a patient
isolate, confirmed (using site-directed mutagenesis) that ?70
improved the fitness of L74V and Q151M so that each double
mutant was more fit than the wild type. The positive impact of
?70 on fitness in this context was also confirmed by studies of
a recombinant virus containing the patient’s viral reverse trans-
criptase sequence (81a).
Serine insertions between codons 69 and 70 of reverse trans-
criptase can also confer multidrug resistance. The replication
fitness of an isolate containing this insertion at codon 69 is
highly dependent on the presence of T215Y (137). These data
indicate that this insertion may not have an adverse effect on
fitness provided that it occurs in combination with another
specific nRTI resistance mutation(s). Such studies also have
the potential to provide information that could assist in de-
signing combination nRTI regimens that delay the onset of
resistance, for example, using nRTIs that select for resistance
mutations that have antagonistic fitness (or resistance) inter-
actions and that avoid those that have synergistic effects to
increase fitness (or resistance).
(ii) Interactions between NNRTI and nRTI resistance mu-
tations. Accumulating data indicate that NNRTI and nRTI resis-
tance mutations can interact to affect viral fitness and that these
interactions may influence how frequently a certain NNRTI-nRTI
resistance mutation combination emerges. For example, the highly
efavirenz-resistant mutant G190E has markedly reduced replication
fitness (82, 127). A study in which the G190E mutant was passaged
in cell culture in the presence of an NNRTI, but without nRTI
selection pressure, demonstrated the acquisition of the nRTI resis-
tance mutation L74V (92). Further studies have demonstrated that
564DYKES AND DEMETERCLIN. MICROBIOL. REV.
the L74V mutation substantially improves the replication fitness of
backbones (82). Studies using the single-cycle Monogram RC assay
also indicated that L74V improved the replication fitness of the
NNRTI-resistant G190S mutant (82).
A recently presented study also demonstrated that the L74V
mutation substantially improves the fitness of the NNRTI-
resistant double mutant K101E/G190S (174a). It was also ob-
served that the NNRTI-resistant double mutant K103N/L100I
was frequently associated with L74V in clinical samples, even
in patients who did not receive didanosine or abacavir at the
time that the triple combination was selected for; the frequent
association of these three mutations was also observed in an-
other study (2, 41a). In cell culture, L74V increased the fitness
of K103N/L100I (94). Thus, it appears that L74V is able to
compensate for the fitness deficits of a number of different
poorly fit, NNRTI-resistant variants. This raises the question
of whether nRTIs that select for L74V, if combined with efa-
virenz, may promote the rapid emergence of highly resistant,
otherwise poorly fit NNRTI-resistant variants. Although this
phenomenon has not been definitively demonstrated, it is very
interesting that a number of the mutants emerging during early
virologic failure of the combination of didanosine, tenofovir,
and efavirenz contained either L74V/G190E or L74V/K103N/
L100I, NNRTI resistance mutations that are normally rare
during therapy with efavirenz (97, 136).
There is a recent report indicating that the TAMs M41L and
T215Y also compensate for the reduced replication fitness of
K101E/G190S, although unlike L74V, these TAMs reduce the
magnitude of efavirenz resistance of the K101E/G190S mutant
(174a). Those studies raise the question of whether interac-
tions between NNRTI and nRTI resistance mutations to affect
viral replication fitness (or resistance) may affect which drug-
resistant mutants emerge or the timing of their emergence.
(iii) Effects of codons in reverse transcriptase not associ-
ated with drug resistance on fitness of nRTI-resistant mu-
tants. As noted above, Q151L, which is likely an intermediate of
the Q151M multi-nRTI resistance mutation, has a profound rep-
lication deficit and was lethal when placed in the reverse trans-
criptase backbone of both HXB-2 and a pretherapy isolate from
a patient that ultimately developed the Q151M variant (64). Of
interest is that the profound replication deficit of Q151L was
markedly improved when placed in the reverse transcriptase
backbone of the posttherapy isolate from this patient. When the
S68G mutation, which was present only in the posttherapy isolate,
was introduced into HXB-2 and pretherapy reverse transcriptase
sequences containing Q151L, the ability of the corresponding
recombinant virus to replicate was partially restored. This may
explain the association between S68G and Q151M observed in
one study of clinical samples (151). This finding also suggests
that sequence context may partially explain the infrequent oc-
currence of Q151M and influence which virus isolates develop
The S68G mutation is also associated with K65R in clinical
samples, although clonal analyses indicate that these mutations
are not always on the same genome (148, 180). A recently
reported study demonstrated that S68G partially compensates
for the fitness deficit of K65R, as does the A62V mutation
As-yet-undefined polymorphisms present in clinical reverse
transcriptase sequences can also modulate the effects of resis-
tance mutations on HIV-1 replication fitness. A double serine
insertion at codon 69 (69insSS), when present in the clinical
reverse transcriptase backbone in which it evolved, conferred
improved replication fitness compared to a laboratory isolate
with the same insertion as well as the same clinical reverse
transcriptase backbone without the insertion (141). A study of
the NNRTI resistance mutations K103N and P236L, which
confer preserved and reduced replication fitness, respectively,
demonstrated that most recombinant viruses containing clini-
cal reverse transcriptase sequences with these mutations rep-
licated as expected in parallel growth kinetic assays (56). How-
ever, there was one reverse transcriptase sequence in which the
relative replication efficiency of K103N and P236L variants
appeared to be reversed, suggesting that polymorphisms in
reverse transcriptase can also modulate the fitness effects of
NNRTI resistance mutations.
Protease. The interactions between major and minor pro-
tease resistance mutations provide clear examples of how the
effect of drug resistance mutations on fitness can be modulated
(see “Minor protease inhibitor resistance mutations” above).
In many instances, the selection for minor mutations after the
emergence of major protease resistance mutations occurs in
association with increases in viral replication fitness, implicat-
ing that compensatory fitness mechanisms are an important
reason for the selection of minor protease resistance mutations
(14, 126). Some, but probably not all, of these mutational
interactions are specific. For example, the reduced fitness of
D30N can be rescued by the accumulation of minor resistance
mutations such as A71V and N88D, whereas the reduced fit-
ness of L90M is rescued by L63P (119, 130). Biochemical
studies also support the ability of M36I and A71V to rescue the
reduced catalytic efficiency of protease containing D30N (30).
There is also evidence that mutations within protease other
than those classified as resistance mutations can modulate the
effects of major protease resistance mutations on viral replica-
tion fitness. For example, D30N is observed much less fre-
quently relative to L90M in subtype C isolates compared with
subtype B. Interestingly, D30N appears to have a greater ad-
verse impact on fitness in a subtype C reverse transcriptase
backbone than in a subtype B backbone, suggesting that re-
duced fitness makes the selection of D30N less likely in a
subtype C background (70, 76).
Patients who fail protease therapy and harbor subtype F
isolates do not acquire D30N and accumulate L90M only in-
frequently (25). This appears to be due to the fact that accu-
mulation of L90M in subtype F is dependent on first acquiring
L89M. Subtype F HIV-1 with L90M alone is highly unfit rel-
ative to subtype F virus containing either L89M/L90M or
L89M alone. These data suggest that the genetic background
can alter protease resistance mutation frequency by influenc-
ing HIV-1 replication fitness.
Envelope. One study has demonstrated that some env-
recombinant viruses derived from patients failing therapy
with enfuvirtide had minimal reductions in fitness, even
though they carried enfuvirtide resistance mutations known
to reduce the replication fitness of HIV-1 (123). This finding
provides another example of how background sequence may
influence a drug resistance mutation’s effect on fitness. Be-
cause of the small number of cloned viruses studied, it is not
VOL. 20, 2007CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS565
clear how often this phenomenon occurs with enfuvirtide-
The replication capacity of some drug-resistant HIV-1 iso-
lates is higher than that of recombinant viruses derived from
laboratory strains that contain the protease and reverse trans-
criptase regions of the corresponding clinical isolates (19, 156).
These findings indicate that protease and reverse transcriptase
sequences do not contain the sole determinants of HIV-1 rep-
lication fitness in drug-resistant isolates and that mutations
outside these regions can modulate the effects of drug resis-
tance mutations on fitness. The following paragraphs describe
specific examples of extragenic compensation.
Protease cleavage site mutations in gag. It has been shown
that the passage of HIV-1 in the presence of a protease inhib-
itor results in drug resistance mutations in protease but also
mutations at the sites where protease cleaves Gag to form p1
and p6 or nucleocapsid (p7) and p1 (55). These cleavage site
mutations improved the efficiency of cleavage by protease in-
hibitor-resistant proteases and were necessary for optimal virus
replication, suggesting that they were selected for because of
their compensatory effects on fitness (55). One study observed
gag cleavage site mutations in HIV-1 isolates from patients
during virologic failure on protease inhibitors, although these
mutations were not present in the majority of protease inhib-
itor-resistant HIV-1 isolates (108). Another study, which fo-
cused on the p7/p1 and p1/p6 cleavage sites, found mutations
in 60% of treatment-experienced patients and in only 10% of
those who were treatment naive (170). Cleavage site mutations
were seen primarily in samples with two or more major resis-
tance mutations in protease, suggesting that cleavage site mu-
tations are selected during therapy (170).
In an analysis of a clinical sequence database, the L449F and
P453L mutations at the p1/p6 cleavage site in Gag were fre-
quently observed in combination with the major protease in-
hibitor resistance mutation I50V, although the two cleavage
site mutations were not observed together in individual clones
derived from patient samples (107). These mutations each
partially improved the replication fitness of HIV-1 strains con-
taining the I50V mutation and increased the degree of pro-
tease inhibitor resistance; no effect on protease inhibitor resis-
tance was seen when these mutations were introduced into a
drug-sensitive laboratory strain (107). In the absence of these
mutations, the I50V mutant accumulated uncleaved p1/p6 pro-
tein; this abnormality was partially corrected by the addition of
one of the cleavage site mutations (107). Those studies also
confirmed that purified protease containing the I50V mutation
cleaved a mutant p1/p6 cleavage site more efficiently than a
wild-type cleavage site (107). Positive and negative associations
between specific cleavage site and major protease-inhibitor
resistance mutations were also observed in another study of
treatment-experienced patients (170).
Other studies of clinical gag and pol sequences also demon-
strated that cleavage site mutations can partially, but not fully,
compensate for the reduced fitness of protease inhibitor-resis-
tant mutants and that these partial improvements in fitness
correlate with partial improvements in the cleavage of the Gag
polyprotein (108). It is possible that there may be evolutionary
barriers to the accumulation of mutations at all cleavage sites.
For example, mutations at the p66/p51 cleavage site dramati-
cally reduced viral fitness and levels of virion-associated re-
verse transcriptase in one study (1).
Another study demonstrated the occurrence of amino acid
insertions near Gag cleavage sites (162). These insertions re-
duced the fitness of drug-sensitive virus but improved the fit-
ness of protease inhibitor-resistant viruses. These differences
in fitness were attributable to changes in cleavage efficiency by
HIV-1 protease (162). Another small study showed that mu-
tations in the cleavage sites between p2 and nucleocapsid and
between p6 and protease can develop in patients on protease
inhibitor therapy and are important for improving the replica-
tion capacity of HIV-1 variants that are resistant to protease
Insertions in gag. The PTAPP motif is a conserved proline-
rich region in the amino terminus of the Gag p6 protein. p6
plays a role in efficient virus release and the incorporation of
Vpr into virions (46). The PTAPP motif is duplicated in some
isolates from heavily nucleoside-experienced patients (83, 96,
114, 131, 162). Infectious clones containing a three-amino-
acid duplication in p6, APPAPP, demonstrated reduced p6
processing and increased packaging of reverse transcriptase
(131). Interestingly, these clones demonstrated a significant
growth advantage over wild-type virus in the presence of nu-
cleoside inhibitors, suggesting that the augmented incor-
poration of reverse transcriptase allowed the virus to become
resistant to nucleoside analogs (131).
In the absence of drug, the fitness of isolates containing
duplications in the PTAPP region without other resistance
mutations is reduced relative to that of the wild type (162).
However, the introduction of these duplications improved the
replication of protease inhibitor-resistant strains (162). Studies
using Western blot analysis of virions have shown that inser-
tions in the PTAPP region impair Gag processing if the pro-
tease sequences contain no drug resistance mutations but im-
prove the processing of protease inhibitor-resistant strains
(162). Presumably, the proximity of these insertions to the p6
cleavage site affects the efficiency of cleavage.
Other mutations in gag. Mutations in gag that were not
associated with cleavage sites or the PTAPP motif were also
observed to accumulate when a laboratory strain was passaged
in the presence of escalating concentrations of protease inhib-
itors (68). Two of these mutations, L75R and H219Q, im-
proved the replication fitness of the protease inhibitor-resis-
tant mutant L10F/V32I/M46I/I54M/A71V/I84V in the absence
of drug (68). These mutations in gag were also essential for the
mutant virus’ replication in the presence of protease inhibitors.
The L75R and H219Q mutations also improved the replication
fitness of the wild-type laboratory strain NL4-3 but did not
allow replication in the presence of protease inhibitors (68). It
is of interest that the H219Q mutation is in the cyclophilin A
binding loop and that the replication advantage conferred by it
was influenced by the cyclophilin A concentration of different
cell types (67), suggesting that gag-cyclophilin A interactions
are important for HIV-1 replication fitness. There are several
additional examples of mutations outside of protease cleavage
sites that improve the fitness of protease inhibitor-resistant
viruses in the absence of cleavage site mutations, indicating
that the coevolution of gag and protease is very complex (124).
566DYKES AND DEMETERCLIN. MICROBIOL. REV.
5?-UTR. An example of extragenic compensation unrelated
to drug resistance is the development of mutations in gag that
compensate for viruses that have deletions in the 5? untrans-
lated region (5?-UTR) (98, 99, 149). The 5?-UTR has several
elements that are important for the HIV-1 viral life cycle (32),
one of which is the dimerization initiation sequence (DIS) (8).
The DIS is important for viral genomic RNA packaging into
the virion because it promotes annealing between the two
RNA genomes and also interacts with the p24 gag protein that
plays a role in the packaging of viral genomes (17). Deletion of
the DIS results in decreased viral infectivity, which can be
restored by mutations in gag (V35I in matrix, I91T in capsid,
T12I in p2, and T24I in nucleocapsid) (98, 99). Another 5?-
UTR element, stem-loop 3, has also been shown to be impor-
tant for genomic RNA packaging and dimerization. The de-
crease in fitness observed by the deletion of this stem-loop can
be compensated for by an A11V mutation in p2 or an I12V
mutation in nucleocapsid (149). It has been shown that dele-
tions in sequences downstream of the DIS can also be com-
pensated for by mutations in the 5?-UTR as well as in nucleo-
capsid and the primer binding site (100). Although this
example does not involve drug resistance mutations, it illus-
trates that compensatory mutations can occur in unexpected
regions of the genome.
Thus, there are substantial data demonstrating that viral
mutations, either within the same gene product (intragenic) or
outside that gene product (extragenic), can modulate the ef-
fects of drug resistance mutations on HIV-1 replication fitness.
Studying the effects of these interactions is important because
one can potentially obtain information on the underlying
mechanisms of fitness compensation and better understand the
interactions between different domains of a drug target or
between different gene products. In addition, if replication
fitness does impact clinical outcome, an understanding of
which mutations modulate the effects of drug resistance mu-
tations on fitness could be relevant to monitoring HIV-1-in-
fected patients failing therapy.
GENETIC DETERMINANTS OF FITNESS
Some studies have evaluated the relative contributions of
different regions of the HIV-1 genome to replication fitness in
cell culture. This information is interesting from a pathogenic
perspective but also provides potentially important data for
designing a clinically relevant recombinant virus assay to assess
the fitness of HIV-1 isolates in different patient populations.
The relative fitnesses of whole-virus isolates of HIV-1 obtained
from untreated patients and recombinant viruses containing
the corresponding env sequences were very similar in a multi-
ple-cycle growth competition assay in PBMCs, suggesting that
env has a dominant influence on the replication fitness of
HIV-1 in untreated patients (116, 142). In one of these studies,
which evaluated two different strains of HIV-1, the relative
fitnesses of the whole virus and its corresponding env-recom-
binant virus also correlated with affinity for CD4 and CCR5
receptors (116). It is interesting that in the one treatment-
experienced patient assessed, the correlation between the rep-
lication of whole virus and that of the env-recombinant virus
was poor, suggesting that resistance mutations in protease and
reverse transcriptase have a greater influence on replication
fitness than env during treatment failure (142). Another study
found that differences in LTRs in different HIV-1 subtypes
were associated with differences in replication fitness that were
cell type specific, suggesting that noncoding regions of the viral
genome may also influence replication fitness (168, 169). A
third study of untreated patients found that the replication
rate, as measured in multiple-cycle, whole-virus, parallel infec-
tions, was significantly associated with fitness as measured by
the Monogram RC assay, although the magnitude of the cor-
relation was only moderate (r2? 0.53; P ? 0.007) (27). This
study suggests that protease and reverse transcriptase se-
quences also contribute to relative fitness.
More studies that directly compare the relative contribu-
tions of different gene segments in a large number of isolates
are needed to definitively determine the relative importance of
different genomic regions in influencing fitness. It does stand to
reason, however, that regions other than protease and reverse
transcriptase could play a dominant role in HIV-1 fitness in
untreated patients, raising questions about the clinical utility of
recombinant virus assays that were designed primarily to assess
drug resistance to measure HIV-1 fitness in this patient pop-
ulation. Although protease and reverse transcriptase se-
quences likely have dominant effects on fitness during virologic
failure, the documented effects of extragenic compensatory
mutations suggest that pol-based recombinant virus assays may
become less predictive of fitness as the duration of virologic
CORRELATION OF HIV-1 REPLICATION FITNESS
WITH CLINICAL OUTCOMES
There is good evidence that HIV-1 replication fitness of a
mutant influences its likelihood of developing during treat-
ment failure. One important question is whether fitness also
influences the likelihood of a mutant being transmitted from
one person to another. Another critical question is whether the
transmission of mutants with reduced fitness leads to improved
clinical outcomes. Since patients are initially infected with one
or a few strains, it is reasonable to postulate that unfit mutants
could persist after the establishment of infection and influence
clinical outcomes. The plasma HIV-1 RNA concentration is a
marker of virus burden and presumably could be influenced by
how efficiently an HIV-1 variant replicates. Thus, it seem rea-
sonable to postulate that initial infection with poorly fit mu-
tants may result in lower viral load set points after primary
infection. Viral load is a major predictor of the rate of CD4?
T-cell decline, and both viral load and CD4?T-cell count are
important predictors of disease progression. Thus, reduced
replication fitness may also be associated with slower disease
progression. Some investigators have postulated that certain
drug-resistant variants affect primarily CD4?T-cell depletion
without substantial effects on viral load. It is not clear how a
reduced rate of replication would selectively affect CD4?T-
cell depletion; therefore, traditional assays for fitness that mea-
sure replication efficiency would likely not be ideal to assess
this aspect of viral fitness, although it is certainly possible that
reduced replication efficiency is one feature of mutants with
VOL. 20, 2007 CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS 567
Mutants with reduced replication fitness can also be selected
for during failure of antiretroviral treatment regimens. Although
these mutants clearly have a relative selection advantage over
wild-type virus in the presence of drug, their absolute rate of
replication in the presence of drug is likely lower than that of the
wild-type virus before initiation of therapy. Therefore, selection
for an unfit variant might result in viral loads during treatment
failure that do not fully return to pretherapy levels. In addition,
since different drug-resistant mutants have different degrees of
impairment in fitness, patients failing similar treatment regimens
could have different viral load and CD4?T-cell trajectories de-
pending on the relative replication fitness of the drug-resistant
mutant that is selected. Evidence for and evidence against an
association between HIV-1 replication fitness and these clinical
outcomes are summarized below.
It should be noted that the establishment of HIV-1 infection
by a given variant is a complex process that is influenced by a
number of factors: the likelihood that transmission from the
source patient will occur, the prevalence of the mutant in
genital secretions, the ability of that mutant to establish infec-
tion in dendritic cells, and the ability of that mutant to replicate
and establish infection in CCR5?/CD4?T cells. Thus, the fact
that a specific mutant occurs less frequently than expected in
recently infected patients may be due to alterations in any or
all of these factors. For the sake of simplicity, we will refer to
this process as “transmission,” recognizing that this term does
not reflect the full complexity of the process by which HIV-1
infection is established.
There is growing evidence that drug-resistant strains of
HIV-1, which usually have impaired replication fitness as mea-
sured in cell culture assays, are underrepresented in primary
HIV-1 infection. Surveillance studies have demonstrated that
approximately 10 to 20% of recently infected patients carry
drug-resistant strains of HIV-1 (74, 101, 157, 183). In addition,
approximately 8 to 10% of drug-resistant isolates are identified
in treatment-naive, chronically infected patients (178). Retro-
spective studies of chronically infected patients that could
serve as potential transmitters of HIV-1 infection indicate that
the prevalence of drug-resistant mutants is substantially higher
than in contemporary cohorts of recently infected patients (24,
182). This evidence has been interpreted to indicate that re-
duced replication fitness correlates with reduced transmission
efficiency, although these studies did not directly measure rep-
There is some evidence that specific drug-resistant mutants
may be less transmissible than others. For example, one study
found that isolates with M184V or major protease inhibitor
resistance mutations were underrepresented in patients with
primary drug-resistant HIV-1 infection compared to patients
with chronic infections (165). In another study, the reverse
transcriptase mutations M184I/V and T215F/Y and the pro-
tease mutations M46I/L were specifically underrepresented in
recently infected patients compared to potential transmitters.
In contrast, V118I, Y181I/C, and K219E/Q in reverse trans-
criptase and I84V and L90M in protease were overrepresented
in recently infected patients and therefore appeared to be
more efficiently transmitted. Other mutations such as Q151M
and Y188C/H/L in reverse transcriptase and D30N, G48V, and
V82A/F/S/T in protease were not detected in any seroconvert-
ers in that study, despite their presence in potential transmit-
None of those studies directly correlated replication fitness,
as measured in a cell culture assay, with likelihood of trans-
mission, so a correlation between replication fitness and trans-
mission efficiency can only be inferred. Some direct support for
this concept is provided by a study that utilized the Monogram
Biosciences RC assay, which measures the impact of the 3? end
of gag, protease, and part of reverse transcriptase on replica-
tion fitness and found that increased fitness was associated with
an increased risk of mother-to-child transmission, even when
accounting for other confounding variables (60).
Consistent with that study is one that found that HIV-1
group O and HIV-2, which occur at a low frequency worldwide
relative to group M HIV-1 isolates, also have reduced replica-
tion fitness relative to group M isolates in a multiple-cycle
whole-virus assay in PBMCs (6). However, another study dem-
onstrated that subtype C, which is the most common subtype
globally, actually has reduced replication fitness compared to
subtype B in PBMCs (6, 11). This finding is not consistent with
the hypothesis that reduced replication fitness in cell culture
correlates with reduced transmission efficiency. Of note is that
some studies suggested that replication in dendritic cells may
be a better predictor of transmission efficiency (50); no differ-
ences between the replication of subtype B and C isolates were
observed in dendritic cells, consistent with this theory (11). The
authors of that study argued that the reduced replication fit-
ness in PBMCs correlates with reduced pathogenicity rather
than transmission efficiency (see below). This property would
theoretically lead to longer asymptomatic stages of clinical
infection and increased opportunities for subtype C transmis-
sions. Clearly, more studies are needed to resolve the question
of whether reduced replication fitness in cell culture correlates
with reduced transmission efficiency and whether replication in
PBMCs is the best in vitro assay to predict transmission effi-
Outcomes in Primary HIV-1 Infection
Evolution of fitness during primary infection. Drug-resistant
mutants selected during therapy in chronically infected pa-
tients are often rapidly overgrown by less drug-resistant vari-
ants once therapy is discontinued. In contrast, once infection
with a drug-resistant variant is established in a newly infected
patient, it can persist as the dominant member of the HIV-1
quasispecies for several years in the absence of drug selection
pressure (21, 22). This finding is likely due to the fact that
initial infection is usually established by a single strain of
HIV-1; therefore, there is presumably no preexisting more-fit,
drug-sensitive mutant during primary infection that can rapidly
emerge when drug selection is removed.
In contrast, significant evolution of HIV-1 at epitopes tar-
geted by cytotoxic T-lymphocyte (CTL) responses has been
observed as early as a few weeks after initial HIV-1 infection
(90, 102). These CTL escape mutants are not as well recog-
nized by the host immune response and indicate that HIV-
specific cellular immune responses exert strong selective pres-
sures on HIV-1 evolution. Development of escape mutants can
568 DYKES AND DEMETERCLIN. MICROBIOL. REV.
also be associated with more rapid disease progression (73).
The primary target of these immune responses appears to be
the viral envelope (102); thus, studies focusing on pol would
not detect these evolutionary changes. It is interesting that not
all potential CTL epitopes recognized by the patient’s immune
response evolve, suggesting that CTL escape mutants at these
epitopes may have a reduced selective advantage for other
reasons (90, 102). The hypothesis that CTL escape mutants
have reduced fitness is supported by studies of simian immu-
nodeficiency virus in which cloned CTL escape mutants ob-
tained from a macaque showed slowed growth in cell culture
and reversion to the CTL-susceptible wild-type sequence after
inoculation into macaques lacking the major histocompatibility
class I determinants necessary for the recognition of the mu-
tant epitopes (62). Studies of escape mutants in the Gag p24
protein demonstrated that these mutations do confer substan-
tial reductions in replication fitness, as measured in a multiple-
cycle cell culture assay (117, 134). It is interesting that these
escape mutants, when they occur, are seen primarily in patients
with HLA types that are associated with the successful control
of HIV viremia. These p24 escape mutants are usually associ-
ated with compensatory mutations in gag (117). Thus, studies
of replication fitness in patients with primary HIV-1 infection,
particularly those who are not infected with drug-resistant vi-
rus or treated with antiretroviral therapy, need to consider the
significant variation and changes in fitness that are occurring in
gag and env.
Impact of HIV-1 replication fitness on CD4?T-cell count
and viral load. A cross-sectional study of 191 acutely and
recently (less than 1 year) HIV-1-infected patients found that
viral replication fitness, using the Monogram RC assay, varied
widely (13). Nineteen percent of isolates demonstrated geno-
typic evidence of resistance to either protease or reverse trans-
criptase inhibitors. There was a statistically significant decrease
in fitness of HIV-1 isolates with mutations conferring resis-
tance to protease inhibitors (P ? 0.01) (Fig. 3). Although
isolates with mutations conferring resistance to nucleoside an-
alogs and NNRTIs tended to have lower replication fitness
than drug-sensitive strains, these differences were not statisti-
cally significant (Fig. 3). Of interest is that only 6% of the
variation in RC values was attributable to drug resistance
This study demonstrated a statistically significant inverse
correlation between replication fitness and CD4?T-cell count
at the first patient evaluation, although the strength of the
correlation was modest (Spearman’s ? of ?0.29; P ? 0.0001)
(Fig. 4). Surprisingly, no correlation was observed between
replication fitness and viral load at baseline (13). Single-step
regression tree analysis demonstrated that a threshold RC
value of 42% best predicted the baseline CD4?T-cell count,
although the 95% confidence intervals were large (12% to
93%). Using this threshold, patients infected with HIV-1 who
had an RC value of ?42% had an average CD4?T-cell count
of 663 cells/?l versus 512 cells/?l for viruses with an RC value
A study of 243 acutely or recently infected patients begin-
ning a combination antiretroviral regimen found no correla-
FIG. 3. Correlation of HIV-1 replication fitness, using the Mono-
gram Biosciences RC assay, with the presence of drug resistance mu-
tations in HIV-1 obtained from antiretroviral-naive patients with early
infection. The y axis represents RC, expressed as a percentage of the
wild-type reference strain. Isolates from 191 patients were placed into
mutually exclusive categories (x axis), depending on whether mutations
conferring resistance to protease inhibitors (PI) with or without other
resistance mutations (PI ? any), nucleoside analogs (nRTI [NRTI])
only, NNRTIs only, or nRTIs plus NNRTIs were present. All isolates
in the “PI ? any” category contained primary (or major) protease
inhibitor resistance mutations. Numbers beneath the graph refer to the
numbers of samples in each category. Only the presence of protease
inhibitor resistance mutations was statistically significantly associated
with RC (P ? 0.01). (Reprinted from reference 13 with permission. ©
2004 by the Infectious Diseases Society of America. All rights re-
FIG. 4. Correlation of HIV-1 replication fitness, using the Mono-
gram Biosciences RC assay, with baseline CD4?T-cell count in anti-
retroviral-naive patients with early infection. The y axis represents the
CD4?T-cell count at baseline, expressed as cells/?l (the y axis label in
the figure is in error); the x axis represents the RC thresholds for each
quartile. There was a statistically significant correlation between re-
duced CD4?T-cell count and RC, which was driven primarily by the
higher CD4?T-cell values in the lowest quartile (Spearman’s ? ?
?0.29; P ? 0.0001). (Reprinted from reference 13 with permission. ©
2004 by the Infectious Diseases Society of America. All rights re-
VOL. 20, 2007 CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS 569
tion between replication fitness measured by the Monogram
RC assay and baseline viral load, baseline CD4?T-cell count,
or viral load responses to antiretroviral therapy (12). Low viral
replication capacity at the time of initiation of therapy did
predict improved CD4?T-cell responses but only after 12
months of therapy (12). The reason for the delayed increase in
the CD4?T-cell count is not clear.
Some isolates obtained during early infection have been
shown to have greater fitness than a wild-type reference strain,
as measured in a whole-virus assay; these properties were not
as pronounced in a recombinant-virus assay using protease and
reverse transcriptase sequences (156). One concerning case
was reported in which a patient was newly infected with a
dual-tropic, multidrug-resistant HIV-1 isolate and had an un-
usually rapid progression to AIDS (113). A pol-recombinant
virus derived from this patient replicated more efficiently than
the wild type in the Monogram Biosciences RC assay (113).
Intact biological clones isolated from this patient also repli-
cated more rapidly than the wild type in growth kinetic assays
and were highly cytopathic in human PBMCs (123a). Although
such rapid clinical progression is uncommon, it has been re-
ported; thus, a cause-and-effect relationship between the in-
creased replication fitness of this isolate and accelerated clin-
ical progression cannot be definitively established.
Outcomes in Chronic HIV-1 Infection
Correlation of HIV-1 replication fitness with clinical outcome
inuntreatedpatientsOne study of HIV-1-infected patients with
unusually prolonged durations of asymptomatic infection
(“long-term nonprogressors”) found reduced viral replication
fitness in some patients, as measured in multiple-cycle, whole-
virus, parallel infections (29). However, patients in this study
also demonstrated evidence of robust HIV-specific cellular
immune responses and high titers of neutralizing antibodies
that could account for their less severe disease (29). Another
study found isolates with reduced HIV-1 replication fitness in
four of seven long-term nonprogressors that persisted over the
course of several years, as measured by a multiple-cycle, whole-
virus, parallel infection assay (18). Replication fitness in this
study also was associated with the plasma HIV-1 RNA con-
centration. Such defects in replication were not observed in
patients with high viral load that did have clinical progression
Another study, which used a multiple-cycle whole-virus
growth competition assay in PBMCs also found a correlation
between replication fitness and disease progression in a small
number of patients (140). Two other studies demonstrated
good correlations between HIV-1 replication fitness, as mea-
sured in multiple-cycle whole-virus parallel infections in
PBMCs, and plasma HIV-1 RNA concentration before the
initiation of antiretroviral therapy (27, 163). In one of those
studies, patients with the CCR5?32 allele, known to have a
beneficial impact on disease progression, or syncytium-induc-
ing virus, which is associated with more rapid disease progres-
sion, were excluded so as to avoid confounding variables in
their analysis (27). Those investigators were unable to find a
correlation between CD8?-mediated cellular immune re-
sponses and plasma HIV-1 RNA concentrations, suggesting
that fitness was the primary influence on the viral load set point
(27). The association between fitness and disease progression
was stronger for the whole-virus assay (r2? 0.71; P ? 0.001)
(Fig. 5A) than for the Monogram RC recombinant virus assay
(r2? 0.44; P ? 0.019) (Fig. 5B), although a significant corre-
lation between viral load and fitness, as measured by the latter
assay, was still observed (27).
A large hemophilia cohort was studied to evaluate the rela-
tionship between HIV-1 replication fitness, as measured using
the Monogram RC assay, and surrogate markers of clinical
outcome (37). Approximately half of the patients were on
therapy, but only five had been treated with a potent combi-
nation antiretroviral regimen. Fitness was significantly corre-
lated with viral load and inversely correlated with baseline
CD4?T-cell counts in the 128 patients who had these assays
performed, although the strength of the association was only
modest (R2value of 0.189 and P value of 0.03 for viral load and
R2value of ?0.199 and P value of 0.02 for CD4?T-cell count)
(Fig. 6a and b, respectively). There was also a trend for repli-
cation fitness to be inversely correlated with change in the
FIG. 5. Correlation of HIV-1 replication fitness, using a whole-
virus parallel infection assay, with baseline plasma HIV-1 RNA con-
centration in chronically infected untreated patients (A) and RC, as
measured by the Monogram Biosciences assay (B). Correlation coef-
ficients (r2) were 0.71 (P ? 0.001) (A) and 0.53 (P ? 0.007) (B). The
solid line indicates the fit of data by linear regression; the dotted lines
indicate the 95% confidence interval for the linear regression. (Re-
printed from reference 27 with permission.)
570DYKES AND DEMETERCLIN. MICROBIOL. REV.
CD4?T-cell count over time when corrected for baseline viral
load and CD4?T-cell count, although the magnitude of the
effect was modest (relative hazard ? 1.07; P ? 0.081). Patients
whose virus had an RC value in the lowest quartile were also at
greater risk for clinical progression to AIDS (Fig. 7).
A retrospective study of 10 patients using a multiple-cycle
whole-virus growth competition assay in PBMCs showed
that viral replication fitness increased over time (164). In-
creases in viral fitness were associated with increasing ge-
netic diversity in env. Syncytium-inducing CXCR4-tropic
(X4) viruses, which develop late in infection and are asso-
ciated with a worse clinical outcome, had greater replication
fitness than non-syncytium-inducing CCR5-tropic (R5) vi-
ruses. However, the increases in fitness that were observed
over time were not due solely to a change in viral coreceptor
Correlation between fitness and outcome of antiretroviral
treatment interruptions. An important observation in HIV-1-
infected patients failing their antiretroviral therapy was that
discontinuing treatment led to further increases in plasma
HIV-1 RNA concentrations and declines in CD4?T-cell
counts, indicating that these apparently failing regimens were
still providing some clinical benefit (41). These changes in viral
load and CD4?T-cell count were also associated with a loss of
drug resistance mutations and a gain in HIV-1 replication
fitness, as measured by the Monogram single-cycle recombi-
nant-virus RC assay, raising the question of whether selection
for a more-fit variant led to rises in plasma viremia (41).
FIG. 6. Correlation of HIV-1 replication fitness, as measured by the Monogram Biosciences RC assay, with baseline CD4?T-cell count (a) and
baseline plasma HIV-1 RNA concentration (b) in a cohort of chronically infected children and adults with hemophilia. (a) y axis, square
root-transformed baseline CD4?T-cell count; x axis, RC expressed as a percentage of the wild-type reference strain. The solid line indicates fit
of data by linear regression (r2? ?0.199; P ? 0.02). (b) y axis, log10-transformed baseline plasma HIV-1 RNA concentration; x axis, RC expressed
as a percentage of the wild-type reference strain. The solid line indicates the fit of data by linear regression (r2? ?0.189; P ? 0.03). (Reprinted
from reference 37 with permission of the publisher.)
VOL. 20, 2007 CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS 571
It is interesting that these rebounds in viremia and declines
in CD4?T-cell count appear to depend on the class of drug
being interrupted. In a nonrandomized study, patients who
selectively interrupted protease inhibitors or NNRTIs had sta-
ble viral loads through 24 weeks of observation, whereas those
who discontinued nucleoside analogs experienced a rapid rise
within 2 weeks of discontinuation (38). Approximately one-
third of patients discontinuing protease inhibitors showed a
loss of protease inhibitor-resistant variants 12 to 36 weeks after
interruption; in these patients, viral fitness increased approxi-
mately twofold after the time of reversion (interquartile range,
1.5 to 4.3) (38). The loss of nucleoside resistance mutations
was incomplete and involved primarily the lamivudine-resis-
tant M184V mutation. However, the loss of M184V occurred
well after the rise in viral load, suggesting that the residual
antiviral activity of nucleosides, rather than an increase in viral
fitness, was the cause of the viral rebound (38). Similar obser-
vations were made in nonrandomized studies of lamivudine or
lamivudine-zidovudine interruption (28, 59). Selective inter-
ruption of the fusion inhibitor enfuvirtide leads to prompt but
limited increases in viral load (?0.1 to 0.2 log10copies/ml),
indicating that this drug also has some limited efficacy despite
the presence of drug-resistant variants (39). These increases in
viral load were not temporally associated with the loss of re-
sistance mutations, suggesting that improvements in viral fit-
ness were not driving these viral rebounds.
Another study found that the fitness of virus (measured
using whole-virus parallel infections in CD8-depleted PBMCs)
before treatment interruption did correlate with the ability to
spontaneously control viremia (P ? 0.014) (163). In that study,
fitness also correlated with the viral load set point before the
initiation of therapy (r2? 0.26; P ? 0.02) but not the change
in viral load (Fig. 8). Thus, the association between fitness and
the ability to control viremia during treatment interruption is
likely due to the association between fitness and pretherapy
viral load levels. Those investigators also found that viral di-
versity, viral replication fitness, and neutralizing antibody ac-
tivity also correlated with the ability to control viremia during
treatment interruption, indicating that a number of confound-
ing variables may complicate the assessment of the role of
replication fitness in outcomes (91). It should be noted that
structured treatment interruptions have not been shown to be
beneficial clinically (16, 58, 150). These observations are inter-
esting in that they provide additional support for a correlation
between replication fitness and viral load in the absence of
Association of HIV-1 replication fitness with antiretroviral
treatment responses. One study of patients failing their anti-
retroviral regimens demonstrated that replication fitness, mea-
sured using the Monogram RC assay, correlated with plasma
HIV-1 viral load (40). Viral replication capacity was also re-
duced in patients who had preserved CD4?T-cell counts de-
spite virologic failure on antiretroviral therapy compared to
patients who experienced CD4?T-cell declines (12% versus
22%; P ? 0.04) (160). More studies are needed to determine
to what extent replication fitness predicts outcome during
treatment failure and whether an assay for replication fitness
could be useful in predicting the viral load and CD4?T-cell
trajectories at the time of first virologic failure. If it was found
useful, such a test might allow more judicious use of antiret-
roviral regimens and preserve future treatment options.
Clinical Predictive Potential of Specific HIV-1 Replication
Based on the studies summarized above, it is difficult to
conclude with certainty which type(s) of fitness assay would be
best suited for future studies of clinical outcome. Certainly, it
FIG. 7. Kaplan-Meier curves for HIV-1 disease progression in chronically infected children and adults with hemophilia according to baseline
HIV-1 replication fitness as measured by the Monogram Biosciences RC assay. The y axis indicates the proportion of patients with AIDS-free
survival; the x axis indicates years after baseline sample. Solid and dashed lines represent the first (RC, ?69%), second (RC, 69 to 94%), third (RC,
95 to 118%), and fourth (RC, ?118%) quartiles of RC values, as labeled in the graph. (Reprinted from reference 37 with permission of the
572DYKES AND DEMETERCLIN. MICROBIOL. REV.
is essential to have an assay with high throughput before sub-
stantial sample sizes can be evaluated. This factor in part
explains the dominance of the Monogram RC assay (a recom-
binant-virus single-cycle assay) in the literature evaluating the
clinical significance of fitness and the smaller sample sizes of
studies in which growth competition or whole-virus assays are
used. Good correlations were found between whole-virus par-
allel assays or a whole-virus growth competition assay and
clinical outcome in untreated patients, suggesting that whole-
virus assays may potentially provide greater clinical predictive
potential than recombinant-virus assays, assuming that the
problem of low throughput could be addressed. This observa-
tion is consistent with the discussion above that regions other
than pol and the 3? end of gag contribute to HIV-1 replication
fitness and are the subject of selective pressures during clinical
infection. Thus, recombinant-virus assays may be more appro-
priate for treatment-experienced patients failing an antiretro-
viral regimen in which major selective forces are acting on pol.
However, none of the assays studied has yet demonstrated a
sufficiently robust association with clinical outcomes that
would warrant routine clinical use at present.
In general, HIV-1 drug-resistant mutants reduce replication
fitness, but different mutants can vary in their degree of fitness
impairment. Fitness, along with the level of drug resistance,
appears to influence the likelihood of a mutant emerging dur-
ing therapy. Fitness is also often associated with how long a
mutant persists in the absence of therapy. There appears to be
a correlation between reduced HIV replication fitness and
either clinical outcome or surrogates of clinical outcome
(CD4?T-cell counts and viral load), although not all studies
found correlations of fitness with both viral load and CD4?
T-cell count. Those studies are retrospective and have not
always accounted for other confounding variables that can
influence outcome. In addition, the larger studies evaluating
the clinical significance of replication fitness have been per-
formed primarily using the Monogram RC assay, which mea-
sures contributions of the 3? end of gag, protease, and part of
reverse transcriptase only. Statistically significant clinical cor-
relations have clearly been found using this assay, but the
modest strength of the associations raises questions as to how
an RC value from an individual patient would be interpreted.
Further study is needed to determine whether measurement of
fitness will be a useful monitoring tool in addition to CD4?
T-cell count, viral load, and resistance testing.
This work was supported in part by R01-AI-041387 and R01-AI-
We thank Hulin Wu for his careful review of the section on math-
ematical approaches to quantifying replication fitness.
1. Abram, M. E., and M. A. Parniak. 2005. Virion instability of human im-
munodeficiency virus type 1 reverse transcriptase (RT) mutated in the
protease cleavage site between RT p51 and the RT RNase H domain.
J. Virol. 79:11952–11961.
2. Ait-Khaled, M., A. Rakik, P. Griffin, C. Stone, N. Richards, D. Thomas, J.
Falloon, M. Tisdale, and the CNA 2007 International Study Team. 2003.
HIV-1 reverse transcriptase and protease resistance mutations selected
during 16–72 weeks of therapy in isolates from antiretroviral therapy-expe-
rienced patients receiving abacavir/efavirenz/amprenavir in the CNA2007
study. Antivir. Ther. 8:111–120.
3. Aquaro, S., V. Svicher, F. Ceccherini-Silberstein, A. Cenci, F. Marcuccilli,
S. Giannella, L. Marcon, R. Calio, J. Balzarini, and C. F. Perno. 2005.
Limited development and progression of resistance of HIV-1 to the nucle-
oside analogue reverse transcriptase inhibitor lamivudine in human primary
macrophages. J. Antimicrob. Chemother. 55:872–878.
4. Archer, R. H., C. Dykes, P. Gerondelis, A. Lloyd, P. Fay, R. C. Reichman,
R. A. Bambara, and L. M. Demeter. 2000. Mutants of human immunode-
ficiency virus type 1 (HIV-1) reverse transcriptase resistant to nonnucleo-
side reverse transcriptase inhibitors demonstrate altered rates of RNase H
cleavage that correlate with HIV-1 replication fitness in cell culture. J. Vi-
5. Archer, R. H., M. Wisniewski, R. A. Bambara, and L. M. Demeter. 2001.
The Y181C mutant of HIV-1 reverse transcriptase resistant to nonnucleo-
side reverse transcriptase inhibitors alters the size distribution of RNase H
cleavages. Biochemistry 40:4087–4095.
6. Arien, K. K., A. Abraha, M. E. Quinones-Mateu, L. Kestens, G. Vanham,
and E. J. Arts. 2005. The replicative fitness of primary human immunode-
ficiency virus type 1 (HIV-1) group M, HIV-1 group O, and HIV-2 isolates.
J. Virol. 79:8979–8990.
7. Armand-Ugon, M., M. E. Quinones-Mateu, A. Gutierez, J. Barretina, J.
Blanco, D. Schols, E. De Clercq, B. Clotet, and J. A. Este. 2003. Reduced
fitness of HIV-1 resistant to CXCR4 antagonists. Antivir. Ther. 8:1–8.
FIG. 8. Association of HIV-1 replication fitness, as measured in a whole-virus parallel infection assay, with ability to control viremia after
treatment interruption. The x axis indicates the slope of the change in culture p24 antigen between days 0 and 6 after infection; the y axis indicates
the log10of patient’s viral load. Panels depict the correlation between HIV-1 replication fitness using this assay and preantiretroviral therapy (ART)
viral load (a), post-structured treatment interruption (STI) viral load (b), and viral load change during STI (c). (Modified from reference 163 with
VOL. 20, 2007CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS 573
8. Awang, G., and D. Sen. 1993. Mode of dimerization of HIV-1 genomic
RNA. Biochemistry 32:11453–11457.
9. Bacheler, L. T., E. D. Anton, P. Kudish, D. Baker, J. Bunville, K. Krakowski,
L. Bolling, M. Aujay, X. V. Wang, D. Ellis, M. F. Becker, A. L. Lasut, H. J.
George, D. R. Spalding, G. Hollis, and K. Abremski. 2000. Human immuno-
deficiency virus type 1 mutations selected in patients failing efavirenz combi-
nation therapy. Antimicrob. Agents Chemother. 44:2475–2484.
10. Back, N. K., M. Nijhuis, W. Keulen, C. A. Boucher, B. O. O. Essink, A. B.
van Kuilenburg, A. H. van Gennip, and B. Berkhout. 1996. Reduced rep-
lication of 3TC-resistant HIV-1 variants in primary cells due to a proces-
sivity defect of the reverse transcriptase enzyme. EMBO J. 15:4040–4049.
11. Ball, S. C., A. Abraha, K. R. Collins, A. J. Marozsan, H. Baird, M. E.
Quinones-Mateu, A. Penn-Nicholson, M. Murray, N. Richard, M. Lobritz,
P. A. Zimmerman, T. Kawamura, A. Blauvelt, and E. J. Arts. 2003. Com-
paring the ex vivo fitness of CCR5-tropic human immunodeficiency virus
type 1 isolates of subtypes B and C. J. Virol. 77:1021–1038.
12. Barbour, J. D., F. M. Hecht, S. J. Little, M. Markowitz, E. S. Daar, A. D.
Kelleher, J. P. Routy, T. B. Campbell, E. S. Rosenberg, M. R. Segal, J.
Weidler, and R. M. Grant. 2006. Greater CD4 T-cell gains after one year of
antiretroviral therapy are associated with lower HIV-1 pol replication ca-
pacity. AIDS 20:2123–2125.
13. Barbour, J. D., F. M. Hecht, T. Wrin, M. R. Segal, C. A. Ramstead, T. J.
Liegler, M. P. Busch, C. J. Petropoulos, N. S. Hellmann, J. O. Kahn, and
R. M. Grant. 2004. Higher CD4?T cell counts associated with low viral pol
replication capacity among treatment-naive adults in early HIV-1 infection.
J. Infect. Dis. 190:251–256.
14. Barbour, J. D., T. Wrin, R. M. Grant, J. N. Martin, M. R. Segal, C. J.
Petropoulos, and S. G. Deeks. 2002. Evolution of phenotypic drug suscep-
tibility and viral replication capacity during long-term virologic failure of
protease inhibitor therapy in human immunodeficiency virus-infected
adults. J. Virol. 76:11104–11112.
15. Bates, M., T. Wrin, W. Huang, C. Petropoulos, and N. Hellmann. 2003.
Practical applications of viral fitness in clinical practice. Curr. Opin. Infect.
16. Benson, C. A., F. Vaida, D. V. Havlir, G. F. Downey, M. M. Lederman, R. M.
Gulick, M. J. Glesby, M. Wantman, C. J. Bixby, A. R. Rinehart, S. Snyder,
R. Wang, S. Patel, and J. W. Mellors. 2006. A randomized trial of treatment
interruption before optimized antiretroviral therapy for persons with drug-
resistant HIV: 48-week virologic results of ACTG A5086. J. Infect. Dis.
17. Berkowitz, R., J. Fisher, and S. P. Goff. 1996. RNA packaging. Curr. Top.
Microbiol. Immunol. 214:177–218.
18. Blaak, H., M. Brouwer, L. J. Ran, F. de Wolf, and H. Schuitemaker. 1998.
In vitro replication kinetics of human immunodeficiency virus type 1
(HIV-1) variants in relation to virus load in long-term survivors of HIV-1
infection. J. Infect. Dis. 177:600–610.
19. Bleiber, G., M. Munoz, A. Ciuffi, P. Meylan, and A. Telenti. 2001. Individual
contributions of mutant protease and reverse transcriptase to viral infec-
tivity, replication, and protein maturation of antiretroviral drug-resistant
human immunodeficiency virus type 1. J. Virol. 75:3291–3300.
20. Boyle, B. A., R. A. Elion, G. J. Moyle, and C. J. Cohen. 2004. Considerations
in selecting protease inhibitor therapy. AIDS Rev. 6:218–225.
21. Brenner, B., J. P. Routy, Y. Quan, D. Moisi, M. Oliveira, D. Turner, M. A.
Wainberg, and Co-Investigators of the Quebec Primary Infection Study.
2004. Persistence of multidrug-resistant HIV-1 in primary infection leading
to superinfection. AIDS 18:1653–1660.
22. Brenner, B. G., J. P. Routy, M. Petrella, D. Moisi, M. Oliveira, M. Detorio,
B. Spira, V. Essabag, B. Conway, R. Lalonde, R. P. Sekaly, and M. A.
Wainberg. 2002. Persistence and fitness of multidrug-resistant human im-
munodeficiency virus type 1 acquired in primary infection. J. Virol. 76:
23. Brown, A. J. L., S. D. Frost, B. Good, E. S. Daar, V. Simon, M. Markowitz,
A. C. Collier, E. Connick, B. Conway, J. B. Margolick, J. P. Routy, J.
Corbeil, N. S. Hellmann, D. D. Richman, and S. J. Little. 2004. Genetic
basis of hypersusceptibility to protease inhibitors and low replicative capac-
ity of human immunodeficiency virus type 1 strains in primary infection.
J. Virol. 78:2242–2246.
24. Brown, A. J. L., S. D. Frost, W. C. Mathews, K. Dawson, N. S. Hellmann,
E. S. Daar, D. D. Richman, and S. J. Little. 2003. Transmission fitness of
drug-resistant human immunodeficiency virus and the prevalence of resis-
tance in the antiretroviral-treated population. J. Infect. Dis. 187:683–686.
25. Calazans, A., R. Brindeiro, P. Brindeiro, H. Verli, M. B. Arruda, L. M.
Gonzalez, J. A. Guimaraes, R. S. Diaz, O. A. Antunes, and A. Tanuri. 2005.
Low accumulation of L90M in protease from subtype F HIV-1 with resis-
tance to protease inhibitors is caused by the L89M polymorphism. J. Infect.
26. Caliendo, A. M., A. Savara, D. An, K. DeVore, J. C. Kaplan, and R. T.
D’Aquila. 1996. Effects of zidovudine-selected human immunodeficiency
virus type 1 reverse transcriptase amino acid substitutions on processive
DNA synthesis and viral replication. J Virol 70:2146–2153.
27. Campbell, T. B., K. Schneider, T. Wrin, C. J. Petropoulos, and E. Connick.
2003. Relationship between in vitro human immunodeficiency virus type 1
replication rate and virus load in plasma. J. Virol. 77:12105–12112.
28. Campbell, T. B., N. S. Shulman, S. C. Johnson, A. R. Zolopa, R. K. Young,
L. Bushman, C. V. Fletcher, E. R. Lanier, T. C. Merigan, and D. R.
Kuritzkes. 2005. Antiviral activity of lamivudine in salvage therapy for
multidrug-resistant HIV-1 infection. Clin. Infect. Dis. 41:236–242.
29. Cao, Y., L. Qin, L. Zhang, J. Safrit, and D. D. Ho. 1995. Virologic and
immunologic characterization of long-term survivors of human immunode-
ficiency virus type 1 infection. N. Engl. J. Med. 332:201–208.
30. Clemente, J. C., R. Hemrajani, L. E. Blum, M. M. Goodenow, and B. M.
Dunn. 2003. Secondary mutations M36I and A71V in the human immuno-
deficiency virus type 1 protease can provide an advantage for the emer-
gence of the primary mutation D30N. Biochemistry 42:15029–15035.
31. Coffin, J. M. 1995. HIV population dynamics in vivo: implications for
genetic variation, pathogenesis, and therapy. Science 267:483–489.
32. Coffin, J. M., S. H. Hughes, and H. E. Varmus. 1997. Retroviruses. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
33. Collins, J. A., M. G. Thompson, E. Paintsil, M. Ricketts, J. Gedzior, and L.
Alexander. 2004. Competitive fitness of nevirapine-resistant human immu-
nodeficiency virus type 1 mutants. J. Virol. 78:603–611.
34. Colonno, R., R. Rose, C. McLaren, A. Thiry, N. Parkin, and J. Friborg.
2004. Identification of I50L as the signature atazanavir (ATV)-resistance
mutation in treatment-naive HIV-1-infected patients receiving ATV-con-
taining regimens. J. Infect. Dis. 189:1802–1810.
35. Cong, M.-E., W. Heneine, and J. G. Garcia-Lerma. 2007. The fitness cost of
mutations associated with human immunodeficiency virus type 1 drug re-
sistance is modulated by mutational interactions. J. Virol. 81:3037–3041.
36. Croteau, G., L. Doyon, D. Thibeault, G. McKercher, L. Pilote, and D.
Lamarre. 1997. Impaired fitness of human immunodeficiency virus type 1
variants with high-level resistance to protease inhibitors. J Virol 71:1089–
37. Daar, E. S., K. L. Kesler, T. Wrin, C. J. Petropoulos, M. Bates, A. Lail, N. S.
Hellmann, E. Gomperts, S. Donfield, and the Hemophilia Growth and
Development Study. 2005. HIV-1 pol replication capacity predicts disease
progression. AIDS 19:871–877.
38. Deeks, S. G., R. Hoh, T. B. Neilands, T. Liegler, F. Aweeka, C. J. Petro-
poulos, R. M. Grant, and J. N. Martin. 2005. Interruption of treatment with
individual therapeutic drug classes in adults with multidrug-resistant HIV-1
infection. J. Infect. Dis. 192:1537–1544.
39. Deeks, S. G., J. Lu, R. Hoh, T. B. Neilands, G. Beatty, W. Huang, T. Liegler,
P. Hunt, J. N. Martin, and D. R. Kuritzkes. 2007. Interruption of enfu-
virtide in HIV-1 infected adults with incomplete viral suppression on an
enfuvirtide-based regimen. J. Infect. Dis. 195:387–391.
E. Hagos, T. Wrin, C. J. Petropoulos, B. Bredt, and J. M. McCune. 2004.
Strong cell-mediated immune responses are associated with the maintenance
of low-level viremia in antiretroviral-treated individuals with drug-resistant
human immunodeficiency virus type 1. J. Infect. Dis. 189:312–321.
41. Deeks, S. G., T. Wrin, T. Liegler, R. Hoh, M. Hayden, J. D. Barbour, N. S.
Hellmann, C. J. Petropoulos, J. M. McCune, M. K. Hellerstein, and R. M.
Grant. 2001. Virologic and immunologic consequences of discontinuing
combination antiretroviral-drug therapy in HIV-infected patients with de-
tectable viremia. N. Engl. J. Med. 344:472–480.
41a.Demeter, L., V. DeGruttola, S. Lustgarten, S. H. Eshleman, S. Hammer, M.
Fischl, and K. Squires. 2004. Abstr. 11th Conf. Retrovir. Opportun. Infect.,
42. Demeter, L. M., H. J. Ribaudo, A. Erice, S. H. Eshleman, S. M. Hammer,
N. S. Hellmann, and M. A. Fischl. 2004. HIV-1 drug resistance in subjects
with advanced HIV-1 infection in whom antiretroviral combination therapy
is failing: a substudy of AIDS Clinical Trials Group Protocol 388. Clin.
Infect. Dis. 39:552–558.
43. Demeter, L. M., R. W. Shafer, P. M. Meehan, J. Holden-Wiltse, M. A.
Fischl, W. W. Freimuth, M. F. Para, and R. C. Reichman. 2000. Delavirdine
susceptibilities and associated reverse transcriptase mutations in human
immunodeficiency virus type 1 isolates from patients in a phase I/II trial of
delavirdine monotherapy (ACTG 260). Antimicrob. Agents Chemother.
44. Department of Health and Human Services. 19 July 2007, accession date.
Guidelines for the use of antiretroviral agents in HIV-1-infected adults and
45. Department of Health and Human Services. 19 July 2007, accession date.
Recommendations for use of antiretroviral drugs in pregnant HIV-1 in-
fected women for maternal health and interventions to reduce perinatal
HIV-1 transmission in the United States. http://www.aidsinfo.nih.gov
46. Dettenhofer, M., and X.-F. Yu. 1999. Proline residues in human immuno-
deficiency virus type 1 p6Gagexert a cell type-dependent effect on viral
replication and virion incorporation of Pol proteins. J. Virol. 73:4696–4704.
47. Deval, J., J. M. Navarro, B. Selmi, J. Courcambeck, J. Boretto, P. Halfon, S.
Garrido-Urbani, J. Sire, and B. Canard. 2004. A loss of viral replicative ca-
pacity correlates with altered DNA polymerization kinetics by the human
574DYKES AND DEMETERCLIN. MICROBIOL. REV.
immunodeficiency virus reverse transcriptase bearing the K65R and L74V
dideoxynucleoside resistance substitutions. J. Biol. Chem. 279:25489–25496.
48. Deval, J., K. L. White, M. D. Miller, N. T. Parkin, J. Courcambeck, P.
Halfon, B. Selmi, J. Boretto, and B. Canard. 2004. Mechanistic basis for
reduced viral and enzymatic fitness of HIV-1 reverse transcriptase contain-
ing both K65R and M184V mutations. J. Biol. Chem. 279:509–516.
49. Diallo, K., B. Marchand, X. Wei, L. Cellai, M. Gotte, and M. A. Wainberg.
2003. Diminished RNA primer usage associated with the L74V and M184V
mutations in the reverse transcriptase of human immunodeficiency virus
type 1 provides a possible mechanism for diminished viral replication ca-
pacity. J. Virol. 77:8621–8632.
50. Dittmar, M. T., G. Simmons, S. Hibbitts, M. O’Hare, S. Louisiri-
rotchanakul, S. Beddows, J. Weber, P. R. Clapham, and R. A. Weiss. 1997.
Langerhans cell tropism of human immunodeficiency virus type 1 subtype A
through F isolates derived from different transmission groups. J. Virol.
51. Domaoal, R. A., R. A. Bambara, and L. M. Demeter. 2006. HIV-1 reverse
transcriptase mutants resistant to nonnucleoside reverse transcriptase in-
hibitors do not adversely affect DNA synthesis: pre-steady-state and steady-
state kinetic studies. J. Acquir. Immune Defic. Syndr. 42:405–411.
52. Domaoal, R. A., and L. M. Demeter. 2004. Structural and biochemical
effects of human immunodeficiency virus mutants resistant to non-nucleo-
side reverse transcriptase inhibitors. Int. J. Biochem. Cell Biol. 36:1735–
53. Domingo, E., E. Martinez-Salas, F. Sobrino, J. C. de la Torre, A. Portela,
J. Ortin, C. Lopez-Galindez, P. Perez-Brena, N. Villanueva, R. Najera, et al.
1985. The quasispecies (extremely heterogeneous) nature of viral RNA
genome populations: biological relevance—a review. Gene 40:1–8.
54. Domingo, E., L. Menendez-Arias, and J. J. Holland. 1997. RNA virus
fitness. Rev. Med. Virol. 7:87–96.
55. Doyon, L., G. Croteau, D. Thibeault, F. Poulin, L. Pilote, and D. Lamarre.
1996. Second locus involved in human immunodeficiency virus type 1 re-
sistance to protease inhibitors. J. Virol. 70:3763–3769.
56. Dykes, C., K. Fox, A. Lloyd, M. Chiulli, E. Morse, and L. M. Demeter. 2001.
Impact of clinical reverse transcriptase sequences on the replication capac-
ity of HIV-1 drug-resistant mutants. Virology 285:193–203.
57. Dykes, C., J. Wang, X. Jin, V. Planelles, D. S. An, A. Tallo, X. Huang, H.
Wu, and L. M. Demeter. 2006. Evaluation of a multiple-cycle, recombinant
virus, growth competition assay that uses flow cytometry to measure repli-
cation efficiency of human immunodeficiency virus type 1 in cell culture.
J. Clin. Microbiol. 44:1930–1943.
58. El-Sadr, W. M., J. D. Lundgren, J. D. Neaton, F. Gordin, D. Abrams, R. C.
Arduino, A. Babiker, W. Burman, N. Clumeck, C. J. Cohen, D. Cohn, D.
Cooper, J. Darbyshire, S. Emery, G. Fatkenheuer, B. Gazzard, B. Grund, J.
Hoy, K. Klingman, M. Losso, N. Markowitz, J. Neuhaus, A. Phillips, and C.
Rappoport. 2006. CD4?count-guided interruption of antiretroviral treat-
ment. N. Engl. J. Med. 355:2283–2296.
59. Eron, J. J., Jr., J. A. Bartlett, J. L. Santana, N. C. Bellos, J. Johnson, A.
Keller, D. R. Kuritzkes, M. H. St. Clair, and V. A. Johnson. 2004. Persistent
antiretroviral activity of nucleoside analogues after prolonged zidovudine
and lamivudine therapy as demonstrated by rapid loss of activity after
discontinuation. J. Acquir. Immune Defic. Syndr. 37:1581–1583.
60. Eshleman, S. H., Y. Lie, D. R. Hoover, S. Chen, S. E. Hudelson, S. A. Fiscus,
C. J. Petropoulos, N. Kumwenda, N. Parkin, and T. E. Taha. 2006. Asso-
ciation between the replication capacity and mother-to-child transmission
of HIV-1, in antiretroviral drug-naive Malawian women. J. Infect. Dis.
61. Frankel, F. A., B. Marchand, D. Turner, M. Gotte, and M. A. Wainberg.
2005. Impaired rescue of chain-terminated DNA synthesis associated with
the L74V mutation in human immunodeficiency virus type 1 reverse trans-
criptase. Antimicrob. Agents Chemother. 49:2657–2664.
62. Friedrich, T. C., E. J. Dodds, L. J. Yant, L. Vojnov, R. Rudersdorf, C.
Cullen, D. T. Evans, R. C. Desrosiers, B. R. Mothe, J. Sidney, A. Sette, K.
Kunstman, S. Wolinsky, M. Piatak, J. Lifson, A. L. Hughes, N. Wilson,
D. H. O’Connor, and D. I. Watkins. 2004. Reversion of CTL escape-variant
immunodeficiency viruses in vivo. Nat. Med. 10:275–281.
63. Gallant, J. E., E. DeJesus, J. R. Arribas, A. L. Pozniak, B. Gazzard, R. E.
Campo, B. Lu, D. McColl, S. Chuck, J. Enejosa, J. J. Toole, and A. K.
Cheng. 2006. Tenofovir DF, emtricitabine, and efavirenz vs. zidovudine,
lamivudine, and efavirenz for HIV. N. Engl. J. Med. 354:251–260.
64. Garcia-Lerma, J. G., P. J. Gerrish, A. C. Wright, S. H. Qari, and W.
Heneine. 2000. Evidence of a role for the Q151L mutation and the viral
background in development of multiple dideoxynucleoside-resistant human
immunodeficiency virus type 1. J. Virol. 74:9339–9346.
65. Garcia-Lerma, J. G., H. MacInnes, D. Bennett, H. Weinstock, and W.
Heneine. 2004. Transmitted human immunodeficiency virus type 1 carrying
the D67N or K219Q/E mutation evolves rapidly to zidovudine resistance in
vitro and shows a high replicative fitness in the presence of zidovudine.
J. Virol. 78:7545–7552.
66. Garcia-Lerma, J. G., S. Nidtha, K. Blumoff, H. Weinstock, and W. Heneine.
2001. Increased ability for selection of zidovudine resistance in a distinct
class of wild-type HIV-1 from drug-naive persons. Proc. Natl. Acad. Sci.
66a.Garcia-Perez, J., M. Perez-Olmeda, S. Sanchez-Palomino, L. Menendez-
Arias, L. Valer, F. Garcia, T. Pumarola, V. Soriano, and J. Alcami. 2006.
Strong decrease in viral replication capacity in HIV-1 containing both
K65R and Q151M mutations. Antivir. Ther. 11:S44.
67. Gatanaga, H., D. Das, Y. Suzuki, D. D. Yeh, K. A. Hussain, A. K. Ghosh,
and H. Mitsuya. 2006. Altered HIV-1 Gag protein interactions with cyclo-
philin A (CypA) on the acquisition of H219Q and H219P substitutions in
the CypA binding loop. J. Biol. Chem. 281:1241–1250.
68. Gatanaga, H., Y. Suzuki, H. Tsang, K. Yoshimura, M. F. Kavlick, K.
Nagashima, R. J. Gorelick, S. Mardy, C. Tang, M. F. Summers, and H.
Mitsuya. 2002. Amino acid substitutions in Gag protein at non-cleavage
sites are indispensable for the development of a high multitude of HIV-1
resistance against protease inhibitors. J. Biol. Chem. 277:5952–5961.
69. Gerondelis, P., R. H. Archer, C. Palaniappan, R. C. Reichman, P. J. Fay,
R. A. Bambara, and L. M. Demeter. 1999. The P236L delavirdine-resistant
human immunodeficiency virus type 1 mutant is replication defective and
demonstrates alterations in both RNA 5?-end- and DNA 3?-end-directed
RNase H activities. J. Virol. 73:5803–5813.
70. Gonzalez, L. M., R. M. Brindeiro, R. S. Aguiar, H. S. Pereira, C. M. Abreu,
M. A. Soares, and A. Tanuri. 2004. Impact of nelfinavir resistance muta-
tions on in vitro phenotype, fitness, and replication capacity of human
immunodeficiency virus type 1 with subtype B and C proteases. Antimicrob.
Agents Chemother. 48:3552–3555.
71. Goudsmit, J., A. de Ronde, E. de Rooij, and R. de Boer. 1997. Broad
spectrum of in vivo fitness of human immunodeficiency virus type 1 sub-
populations differing at reverse transcriptase codons 41 and 215. J. Virol.
72. Goudsmit, J., A. De Ronde, D. D. Ho, and A. S. Perelson. 1996. Human
immunodeficiency virus fitness in vivo: calculations based on a single
zidovudine resistance mutation at codon 215 of reverse transcriptase. J. Vi-
73. Goulder, P. J., R. E. Phillips, R. A. Colbert, S. McAdam, G. Ogg, M. A.
Nowak, P. Giangrande, G. Luzzi, B. Morgan, A. Edwards, A. J. McMichael,
and S. Rowland-Jones. 1997. Late escape from an immunodominant cyto-
toxic T-lymphocyte response associated with progression to AIDS. Nat.
74. Grant, R. M., F. M. Hecht, M. Warmerdam, L. Liu, T. Liegler, C. J.
Petropoulos, N. S. Hellmann, M. Chesney, M. P. Busch, and J. O. Kahn.
2002. Time trends in primary HIV-1 drug resistance among recently in-
fected persons. JAMA 288:181–188.
75. Greenberg, M., N. Cammack, M. Salgo, and L. Smiley. 2004. HIV fusion
and its inhibition in antiretroviral therapy. Rev. Med. Virol. 14:321–337.
76. Grossman, Z., E. E. Paxinos, D. Averbuch, S. Maayan, N. T. Parkin, D.
Engelhard, M. Lorber, V. Istomin, Y. Shaked, E. Mendelson, D. Ram, C. J.
Petropoulos, and J. M. Schapiro. 2004. Mutation D30N is not preferentially
selected by human immunodeficiency virus type 1 subtype C in the devel-
opment of resistance to nelfinavir. Antimicrob. Agents Chemother. 48:
77. Hanna, G. J., V. A. Johnson, D. R. Kuritzkes, D. D. Richman, A. J. Brown,
A. V. Savara, J. D. Hazelwood, and R. T. D’Aquila. 2000. Patterns of
resistance mutations selected by treatment of human immunodeficiency
virus type 1 infection with zidovudine, didanosine, and nevirapine. J. Infect.
78. Harrigan, P. R., S. Bloor, and B. A. Larder. 1998. Relative replicative
fitness of zidovudine-resistant human immunodeficiency virus type 1 iso-
lates in vitro. J. Virol. 72:3773–3778.
78a.Henderson, G. J., N. Parkin, and R. Swanstrom. 2005. A systematic analysis
of the fitness effects of mutations associated with resistance to protease
inhibitors. Antivir. Ther. 10:S165.
79. Ho, D. D., A. U. Neumann, A. S. Perelson, W. Chen, J. M. Leonard, and M.
Markowitz. 1995. Rapid turnover of plasma virions and CD4 lymphocytes
in HIV-1 infection. Nature 373:123–126.
80. Hu, W. S., and H. M. Temin. 1990. Genetic consequences of packaging two
RNA genomes in one retroviral particle: pseudodiploidy and high rate of
genetic recombination. Proc. Natl. Acad. Sci. USA 87:1556–1560.
81. Hu, Z., F. Giguel, H. Hatano, P. Reid, J. Lu, and D. R. Kuritzkes. 2006.
Fitness comparison of thymidine analog resistance pathways in human
immunodeficiency virus type 1. J. Virol. 80:7020–7027.
81a.Hu, Z. X., H. Hatano, M. Wild, R. Kalayjian, B. Gripshover, and D. R.
Kuritzkes. 2005. Relative fitness and infectivity of a clinical HIV-1 isolate
with a deletion of codon 70 in reverse transcriptase. Antivir. Ther. 10:S178.
82. Huang, W., A. Gamarnik, K. Limoli, C. J. Petropoulos, and J. M. Whit-
comb. 2003. Amino acid substitutions at position 190 of human immuno-
deficiency virus type 1 reverse transcriptase increase susceptibility to dela-
virdine and impair virus replication. J. Virol. 77:1512–1523.
83. Ibe, S., N. Shibata, M. Utsumi, and T. Kaneda. 2003. Selection of human
immunodeficiency virus type 1 variants with an insertion mutation in the
p6(gag) and p6(pol) genes under highly active antiretroviral therapy. Mi-
crobiol. Immunol. 47:71–79.
84. Iglesias-Ussel, M. D., C. Casado, E. Yuste, I. Olivares, and C. Lopez-
VOL. 20, 2007CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS575
Galindez. 2002. In vitro analysis of human immunodeficiency virus type 1
resistance to nevirapine and fitness determination of resistant variants.
J. Gen. Virol. 83:93–101.
85. Imamichi, T. 2004. Action of anti-HIV drugs and resistance: reverse trans-
criptase inhibitors and protease inhibitors. Curr. Pharm. Des. 10:4039–
86. Imamichi, T., S. C. Berg, H. Imamichi, J. C. Lopez, J. A. Metcalf, J. Falloon,
and H. C. Lane. 2000. Relative replication fitness of a high-level 3?-azido-
3?-deoxythymidine-resistant variant of human immunodeficiency virus type
1 possessing an amino acid deletion at codon 67 and a novel substitution
(Thr3Gly) at codon 69. J. Virol. 74:10958–10964.
87. Johnson, V. A., F. Brun-Vezinet, B. Clotet, D. Kuritzkes, D. Pillay, J. M.
Schapiro, and D. D. Richman. 2006. Update of the drug resistance muta-
tions in HIV-1: Fall 2006. Top. HIV Med. 14:125–130.
88. Joly, V., D. Descamps, G. Peytavin, F. Touati, F. Mentre, X. Duval, S.
Delarue, P. Yeni, and F. Brun-Vezinet. 2004. Evolution of human immu-
nodeficiency virus type 1 (HIV-1) resistance mutations in nonnucleoside
reverse transcriptase inhibitors (NNRTIs) in HIV-1-infected patients
switched to antiretroviral therapy without NNRTIs. Antimicrob. Agents
89. Joly, V., and P. Yeni. 1999. Non nucleoside reverse transcriptase inhibitors.
AIDS Rev. 1:37–44.
90. Jones, N. A., X. Wei, D. R. Flower, M. Wong, F. Michor, M. S. Saag, B. H.
Hahn, M. A. Nowak, G. M. Shaw, and P. Borrow. 2004. Determinants of
human immunodeficiency virus type 1 escape from the primary CD8?
cytotoxic T lymphocyte response. J. Exp. Med. 200:1243–1256.
91. Joos, B., A. Trkola, M. Fischer, H. Kuster, P. Rusert, C. Leemann, J. Boni,
A. Oxenius, D. A. Price, R. E. Phillips, J. K. Wong, B. Hirschel, R. Weber,
and H. F. Gunthard. 2005. Low human immunodeficiency virus envelope
diversity correlates with low in vitro replication capacity and predicts spon-
taneous control of plasma viremia after treatment interruptions. J. Virol.
91a.Kitchen, C., P. Krogstad, and S. Kitchen. 2006. Abstr. 13th Conf. Retrovir.
Opportun. Infect., abstr. 626.
92. Kleim, J. P., M. Rosner, I. Winkler, A. Paessens, R. Kirsch, Y. Hsiou, E.
Arnold, and G. Riess. 1996. Selective pressure of a quinoxaline nonnucleo-
side inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse
transcriptase (RT) on HIV-1 replication results in the emergence of nucle-
oside RT-inhibitor-specific (RT Leu-743Val or Ile and Val-753Leu or
Ile) HIV-1 mutants. Proc. Natl. Acad. Sci. USA 93:34–38.
92a.Koch, S., R. Coman, I. Munoz, B. M. Dunn, J. W. Sleasman, and M. M.
Doodenow. 2006. Amino acid changes in Gag that develop in vivo during
protease inhibitor therapy have a dominant effect on viral replication and
sensitivity to protease inhibitors ex vivo. Antivir. Ther. 11:S66.
93. Kosalaraksa, P., M. F. Kavlick, V. Maroun, R. Le, and H. Mitsuya. 1999.
Comparative fitness of multi-dideoxynucleoside-resistant human immuno-
deficiency virus type 1 (HIV-1) in an in vitro competitive HIV-1 replication
assay. J. Virol. 73:5356–5363.
94. Koval, C. E., C. Dykes, J. Wang, and L. M. Demeter. 2006. Relative repli-
cation fitness of efavirenz-resistant mutants of HIV-1: correlation with
frequency during clinical therapy and evidence of compensation for the
reduced fitness of K103N ? L100I by the nucleoside resistance mutation
L74V. Virology 353:184–192.
95. Kozal, M. J., N. Shah, N. Shen, R. Yang, R. Fucini, T. C. Merigan, D. D.
Richman, D. Morris, E. Hubbell, M. Chee, and T. R. Gingeras. 1996.
Extensive polymorphisms observed in HIV-1 clade B protease gene using
high-density oligonucleotide arrays. Nat. Med. 2:753–759.
96. Lastere, S., C. Dalban, G. Collin, D. Descamps, P. M. Girard, F. Clavel, D.
Costagliola, F. Brun-Vezinet, and the NARVAL Trial Group. 2004. Impact
of insertions in the HIV-1 p6 PTAPP region on the virological response to
amprenavir. Antivir. Ther. 9:221–227.
97. Leon, A., E. Martinez, J. Mallolas, M. Laguno, J. L. Blanco, T. Pumarola,
and J. M. Gatell. 2005. Early virological failure in treatment-naive HIV-
infected adults receiving didanosine and tenofovir plus efavirenz or nevi-
rapine. AIDS 19:213–215.
98. Liang, C., L. Rong, M. Laughrea, L. Kleiman, and M. A. Wainberg. 1998.
Compensatory point mutations in the human immunodeficiency virus type
1 Gag region that are distal from deletion mutations in the dimerization
initiation site can restore viral replication. J. Virol. 72:6629–6636.
99. Liang, C., L. Rong, Y. Quan, M. Laughrea, L. Kleiman, and M. A. Wain-
berg. 1999. Mutations within four distinct Gag proteins are required to
restore replication of human immunodeficiency virus type 1 after deletion
mutagenesis within the dimerization initiation site. J. Virol. 73:7014–7020.
100. Liang, C., L. Rong, R. S. Russell, and M. A. Wainberg. 2000. Deletion
mutagenesis downstream of the 5? long terminal repeat of human immu-
nodeficiency virus type 1 is compensated for by point mutations in both the
U5 region and gag gene. J. Virol. 74:6251–6261.
101. Little, S. J., S. Holte, J. P. Routy, E. S. Daar, M. Markowitz, A. C. Collier,
R. A. Koup, J. W. Mellors, E. Connick, B. Conway, M. Kilby, L. Wang, J. M.
Whitcomb, N. S. Hellmann, and D. D. Richman. 2002. Antiretroviral-drug
resistance among patients recently infected with HIV. N. Engl. J. Med.
102. Liu, Y., J. McNevin, J. Cao, H. Zhao, I. Genowati, K. Wong, S. McLaughlin,
M. D. McSweyn, K. Diem, C. E. Stevens, J. Maenza, H. He, D. C. Nickle, D.
Shriner, S. E. Holte, A. C. Collier, L. Corey, M. J. McElrath, and J. I.
Mullins. 2006. Selection on the human immunodeficiency virus type 1
proteome following primary infection. J. Virol. 80:9519–9529.
103. Lu, J., and D. R. Kuritzkes. 2001. A novel recombinant marker virus assay
for comparing the relative fitness of HIV-1 reverse transcriptase variants. J.
Acquir. Immune Defic. Syndr. 27:7–13.
104. Lu, J., P. Sista, F. Giguel, M. Greenberg, and D. R. Kuritzkes. 2004.
Relative replicative fitness of human immunodeficiency virus type 1 mu-
tants resistant to enfuvirtide (T-20). J. Virol. 78:4628–4637.
105. Lu, J., J. Whitcomb, and D. R. Kuritzkes. 2005. Effect of the Q207D
mutation in HIV type 1 reverse transcriptase on zidovudine susceptibility
and replicative fitness. J. Acquir. Immune Defic. Syndr. 40:20–23.
106. Maeda, Y., D. J. Venzon, and H. Mitsuya. 1998. Altered drug sensitivity,
fitness, and evolution of human immunodeficiency virus type 1 with pol
gene mutations conferring multi-dideoxynucleoside resistance. J. Infect.
107. Maguire, M. F., R. Guinea, P. Griffin, S. Macmanus, R. C. Elston, J.
Wolfram, N. Richards, M. H. Hanlon, D. J. Porter, T. Wrin, N. Parkin, M.
Tisdale, E. Furfine, C. Petropoulos, B. W. Snowden, and J. P. Kleim. 2002.
Changes in human immunodeficiency virus type 1 Gag at positions L449
and P453 are linked to I50V protease mutants in vivo and cause reduction
of sensitivity to amprenavir and improved viral fitness in vitro. J. Virol.
108. Mammano, F., C. Petit, and F. Clavel. 1998. Resistance-associated loss of
viral fitness in human immunodeficiency virus type 1: phenotypic analysis of
protease and gag coevolution in protease inhibitor-treated patients. J. Virol.
109. Mammano, F., V. Trouplin, V. Zennou, and F. Clavel. 2000. Retracing the
evolutionary pathways of human immunodeficiency virus type 1 resistance
to protease inhibitors: virus fitness in the absence and in the presence of
drug. J. Virol. 74:8524–8531.
110. Mansky, L. M., and H. M. Temin. 1995. Lower in vivo mutation rate of
human immunodeficiency virus type 1 than that predicted from the fidelity
of purified reverse transcriptase. J. Virol. 69:5087–5094.
111. Marcelin, A. G., C. Delaugerre, M. Wirden, P. Viegas, A. Simon, C. Katlama,
and V. Calvez. 2004. Thymidine analogue reverse transcriptase inhibitors re-
sistance mutations profiles and association to other nucleoside reverse trans-
criptase inhibitors resistance mutations observed in the context of virological
failure. J. Med. Virol. 72:162–165.
112. Maree, A. F., W. Keulen, C. A. Boucher, and R. J. De Boer. 2000. Estimating
relative fitness in viral competition experiments. J. Virol. 74:11067–11072.
113. Markowitz, M., H. Mohri, S. Mehandru, A. Shet, L. Berry, R. Kalyanara-
man, A. Kim, C. Chung, P. Jean-Pierre, A. Horowitz, M. La Mar, T. Wrin,
N. Parkin, M. Poles, C. Petropoulos, M. Mullen, D. Boden, and D. D. Ho.
2005. Infection with multidrug resistant, dual-tropic HIV-1 and rapid pro-
gression to AIDS: a case report. Lancet 365:1031–1038.
114. Marlowe, N., T. Flys, J. Hackett, Jr., M. Schumaker, J. B. Jackson, S. H.
Eshleman, the HIV Prevention Trials Network, and the Adult AIDS Clin-
ical Trials Group. 2004. Analysis of insertions and deletions in the gag p6
region of diverse HIV type 1 strains. AIDS Res. Hum. Retrovir. 20:1119–
115. Marozsan, A. J., E. Fraundorf, A. Abraha, H. Baird, D. Moore, R. Troyer,
I. Nankja, and E. J. Arts. 2004. Relationships between infectious titer,
capsid protein levels, and reverse transcriptase activities of diverse human
immunodeficiency virus type 1 isolates. J. Virol. 78:11130–11141.
116. Marozsan, A. J., D. M. Moore, M. A. Lobritz, E. Fraundorf, A. Abraha, J. D.
Reeves, and E. J. Arts. 2005. Differences in the fitness of two diverse
wild-type human immunodeficiency virus type 1 isolates are related to the
efficiency of cell binding and entry. J. Virol. 79:7121–7134.
117. Martinez-Picado, J., J. G. Prado, E. E. Fry, K. Pfafferott, A. Leslie, S.
Chetty, C. Thobakgale, I. Honeyborne, H. Crawford, P. Matthews, T. Pillay,
C. Rousseau, J. I. Mullins, C. Brander, B. D. Walker, D. I. Stuart, P.
Kiepiela, and P. Goulder. 2006. Fitness cost of escape mutations in p24 Gag
in association with control of human immunodeficiency virus type 1. J. Vi-
118. Martinez-Picado, J., A. V. Savara, L. Shi, L. Sutton, and R. T. D’Aquila.
2000. Fitness of human immunodeficiency virus type 1 protease inhibitor-
selected single mutants. Virology 275:318–322.
119. Martinez-Picado, J., A. V. Savara, L. Sutton, and R. T. D’Aquila. 1999.
Replicative fitness of protease inhibitor-resistant mutants of human immu-
nodeficiency virus type 1. J. Virol. 73:3744–3752.
120. Martinez-Picado, J., T. Wrin, S. D. Frost, B. Clotet, L. Ruiz, A. J. Brown,
C. J. Petropoulos, and N. T. Parkin. 2005. Phenotypic hypersusceptibility to
multiple protease inhibitors and low replicative capacity in patients who are
chronically infected with human immunodeficiency virus type 1. J. Virol.
121. Melby, T., P. Sista, R. DeMasi, T. Kirkland, N. Roberts, M. Salgo, G.
Heilek-Snyder, N. Cammack, T. J. Matthews, and M. L. Greenberg. 2006.
Characterization of envelope glycoprotein gp41 genotype and phenotypic
susceptibility to enfuvirtide at baseline and on treatment in the phase III
576DYKES AND DEMETERCLIN. MICROBIOL. REV.
clinical trials TORO-1 and TORO-2. AIDS Res. Hum. Retrovir. 22:375–
122. Menzo, S., A. Castagna, A. Monachetti, H. Hasson, A. Danise, E. Carini, P.
Bagnarelli, A. Lazzarin, and M. Clementi. 2004. Genotype and phenotype
patterns of human immunodeficiency virus type 1 resistance to enfuvirtide
during long-term treatment. Antimicrob. Agents Chemother. 48:3253–3259.
123. Menzo, S., A. Castagna, A. Monachetti, H. Hasson, A. Danise, E. Carini, P.
Bagnarelli, A. Lazzarin, and M. Clementi. 2004. Resistance and replicative
capacity of HIV-1 strains selected in vivo by long-term enfuvirtide treat-
ment. New Microbiol. 27:51–61.
123a.Mohri, H., P. Jean-Pierre, L. Berry, A. Kim, C. Chung, V. Manuelli, D.
Boden, S. Mehandru, A. Shet, and M. Markowitz. 2006. Abstr. 13th Conf.
Retrovir. Opportun. Infect., abstr. 625.
124. Myint, L., M. Matsuda, Z. Matsuda, Y. Yokomaku, T. Chiba, A. Okano, K.
Yamada, and W. Sugiura. 2004. Gag non-cleavage site mutations contribute
to full recovery of viral fitness in protease inhibitor-resistant human immu-
nodeficiency virus type 1. Antimicrob. Agents Chemother. 48:444–452.
125. Neumann, T., I. Hagmann, S. Lohrengel, M. L. Heil, C. A. Derdeyn, H. G.
Krausslich, and M. T. Dittmar. 2005. T20-insensitive HIV-1 from naive
patients exhibits high viral fitness in a novel dual-color competition assay on
primary cells. Virology 333:251–262.
126. Nijhuis, M., R. Schuurman, D. de Jong, J. Erickson, E. Gustchina, J.
Albert, P. Schipper, S. Gulnik, and C. A. Boucher. 1999. Increased fitness
of drug resistant HIV-1 protease as a result of acquisition of compensatory
mutations during suboptimal therapy. AIDS 13:2349–2359.
127. Olmsted, R. A., D. E. Slade, L. A. Kopta, S. M. Poppe, T. J. Poel, S. W.
Newport, K. B. Rank, C. Biles, R. A. Morge, T. J. Dueweke, Y. Yagi, D. L.
Romero, R. C. Thomas, S. K. Sharma, and W. G. Tarpley. 1996. (Alkyl-
amino)piperidine bis(heteroaryl)piperizine analogs are potent, broad-spec-
trum nonnucleoside reverse transcriptase inhibitors of drug-resistant iso-
lates of human immunodeficiency virus type 1 (HIV-1) and select for
drug-resistant variants of HIV-1IIIBwith reduced replication phenotypes.
J. Virol. 70:3698–3705.
128. Patick, A. K., M. Duran, Y. Cao, D. Shugarts, M. R. Keller, E. Mazabel, M.
Knowles, S. Chapman, D. R. Kuritzkes, and M. Markowitz. 1998. Geno-
typic and phenotypic characterization of human immunodeficiency virus
type 1 variants isolated from patients treated with the protease inhibitor
nelfinavir. Antimicrob. Agents Chemother. 42:2637–2644.
129. Perez-Bercoff, D., S. Wurtzer, S. Compain, H. Benech, and F. Clavel. 2007.
Human immunodeficiency virus type 1 resistance to nucleoside analogues
and replicative capacity in primary human macrophages. J. Virol. 81:4540–
130. Perrin, V., and F. Mammano. 2003. Parameters driving the selection of
nelfinavir-resistant human immunodeficiency virus type 1 variants. J. Virol.
131. Peters, S., M. Munoz, S. Yerly, V. Sanchez-Merino, C. Lopez-Galindez, L.
Perrin, B. Larder, D. Cmarko, S. Fakan, P. Meylan, and A. Telenti. 2001.
Resistance to nucleoside analog reverse transcriptase inhibitors mediated
by human immunodeficiency virus type 1 p6 protein. J. Virol. 75:9644–9653.
132. Petrella, M., and M. A. Wainberg. 2002. Might the M184V substitution in
HIV-1 RT confer clinical benefit? AIDS Rev. 4:224–232.
133. Petropoulos, C. J., N. T. Parkin, K. L. Limoli, Y. S. Lie, T. Wrin, W. Huang,
H. Tian, D. Smith, G. A. Winslow, D. J. Capon, and J. M. Whitcomb. 2000.
A novel phenotypic drug susceptibility assay for human immunodeficiency
virus type 1. Antimicrob. Agents Chemother. 44:920–928.
134. Peyerl, F. W., H. S. Bazick, M. H. Newberg, D. H. Barouch, J. Sodroski, and
N. L. Letvin. 2004. Fitness costs limit viral escape from cytotoxic T lym-
phocytes at a structurally constrained epitope. J. Virol. 78:13901–13910.
135. Picchio, G. R., H. Valdez, R. Sabbe, A. L. Landay, D. R. Kuritzkes, M. M.
Lederman, and D. E. Mosier. 2000. Altered viral fitness of HIV-1 following
failure of protease inhibitor-based therapy. J. Acquir. Immune Defic.
136. Podzamczer, D., E. Ferrer, J. M. Gatell, J. Niubo, D. Dalmau, A. Leon, H.
Knobel, C. Polo, D. Iniguez, and I. Ruiz. 2005. Early virological failure with
a combination of tenofovir, didanosine and efavirenz. Antivir. Ther. 10:
137. Prado, J. G., S. Franco, T. Matamoros, L. Ruiz, B. Clotet, L. Menendez-
Arias, M. A. Martinez, and J. Martinez-Picado. 2004. Relative replication
fitness of multi-nucleoside analogue-resistant HIV-1 strains bearing a
dipeptide insertion in the fingers subdomain of the reverse transcriptase
and mutations at codons 67 and 215. Virology 326:103–112.
138. Prado, J. G., T. Wrin, J. Beauchaine, L. Ruiz, C. J. Petropoulos, S. D. Frost,
B. Clotet, R. T. D’Aquila, and J. Martinez-Picado. 2002. Amprenavir-
resistant HIV-1 exhibits lopinavir cross-resistance and reduced replication
capacity. AIDS 16:1009–1017.
139. Quan, Y., B. G. Brenner, M. Oliveira, and M. A. Wainberg. 2003. Lamivu-
dine can exert a modest antiviral effect against human immunodeficiency
virus type 1 containing the M184V mutation. Antimicrob. Agents Che-
140. Quin ˜ones-Mateu, M. E., S. C. Ball, A. J. Marozsan, V. S. Torre, J. L.
Albright, G. Vanham, G. van Der Groen, R. L. Colebunders, and E. J. Arts.
2000. A dual infection/competition assay shows a correlation between ex
vivo human immunodeficiency virus type 1 fitness and disease progression.
J. Virol. 74:9222–9233.
141. Quin ˜ones-Mateu, M. E., M. Tadele, M. Parera, A. Mas, J. Weber, H. R.
Rangel, B. Chakraborty, B. Clotet, E. Domingo, L. Mene ´ndez-Arias, and
M. A. Martı ´nez. 2002. Insertions in the reverse transcriptase increase both
drug resistance and viral fitness in a human immunodeficiency virus type 1
isolate harboring the multi-nucleoside reverse transcriptase inhibitor resis-
tance 69 insertion complex mutation. J. Virol. 76:10546–10552.
142. Rangel, H. R., J. Weber, B. Chakraborty, A. Gutierrez, M. L. Marotta, M.
Mirza, P. Kiser, M. A. Martinez, J. A. Este, and M. E. Quinones-Mateu.
2003. Role of the human immunodeficiency virus type 1 envelope gene in
viral fitness. J. Virol. 77:9069–9073.
143. Reeves, J. D., F. H. Lee, J. L. Miamidian, C. B. Jabara, M. M. Juntilla, and
R. W. Doms. 2005. Enfuvirtide resistance mutations: impact on human
immunodeficiency virus envelope function, entry inhibitor sensitivity, and
virus neutralization. J. Virol. 79:4991–4999.
144. Resch, W., R. Ziermann, N. Parkin, A. Gamarnik, and R. Swanstrom. 2002.
Nelfinavir-resistant, amprenavir-hypersusceptible strains of human immu-
nodeficiency virus type 1 carrying an N88S mutation in protease have
reduced infectivity, reduced replication capacity, and reduced fitness and
process the Gag polyprotein precursor aberrantly. J. Virol. 76:8659–8666.
145. Richman, D. D., D. Havlir, J. Corbeil, D. Looney, C. Ignacio, S. A. Spector,
J. Sullivan, S. Cheeseman, K. Barringer, and D. Pauletti. 1994. Nevirapine
resistance mutations of human immunodeficiency virus type 1 selected
during therapy. J. Virol. 68:1660–1666.
146. Rimsky, L. T., D. C. Shugars, and T. J. Matthews. 1998. Determinants of
human immunodeficiency virus type 1 resistance to gp41-derived inhibitory
peptides. J. Virol. 72:986–993.
146a.Rimsky, L. T., H. van Marck, B. Maes, Y. Verlinden, L. Bacheler, H. Azjin,
I. De Baere, G. Kraus, and M-P. de Be ´thune. 2006. A novel assay to
determine the HIV-1 in vitro replication rate independent of virus input
concentration, and its application to analyze the evolution of the replication
rate of viruses emerging from in vitro selection with TMC114. Antivir. Ther.
147. Roberts, J. D., K. Bebenek, and T. A. Kunkel. 1988. The accuracy of reverse
transcriptase from HIV-1. Science 242:1171–1173.
148. Roge, B. T., T. L. Katzenstein, N. Obel, H. Nielsen, O. Kirk, C. Pedersen,
L. Mathiesen, J. Lundgren, and J. Gerstoft. 2003. K65R with and without
S68: a new resistance profile in vivo detected in most patients failing
abacavir, didanosine and stavudine. Antivir. Ther. 8:173–182.
149. Rong, L., R. S. Russell, J. Hu, M. Laughrea, M. A. Wainberg, and C. Liang.
2003. Deletion of stem-loop 3 is compensated by second-site mutations
within the Gag protein of human immunodeficiency virus type 1. Virology
150. Ruiz, L., R. Paredes, G. Gomez, J. Romeu, P. Domingo, N. Perez-Alvarez,
G. Tambussi, J. M. Llibre, J. Martinez-Picado, F. Vidal, C. R. Fumaz, and
B. Clotet. 2007. Antiretroviral therapy interruption guided by CD4 cell
counts and plasma HIV-1 RNA levels in chronically HIV-1-infected pa-
tients. AIDS 21:169–178.
151. Schmit, J. C., K. Van Laethem, L. Ruiz, P. Hermans, S. Sprecher, A.
Sonnerborg, M. Leal, T. Harrer, B. Clotet, V. Arendt, E. Lissen, M.
Witvrouw, J. Desmyter, E. De Clercq, and A. M. Vandamme. 1998. Multiple
dideoxynucleoside analogue-resistant (MddNR) HIV-1 strains isolated
from patients from different European countries. AIDS 12:2007–2015.
152. Schock, H. B., V. M. Garsky, and L. C. Kuo. 1996. Mutational anatomy of
an HIV-1 protease variant conferring cross-resistance to protease inhibitors
in clinical trials. Compensatory modulations of binding and activity. J. Biol.
153. Schuurman, R., M. Nijhuis, R. van Leeuwen, P. Schipper, D. de Jong, P.
Collis, S. A. Danner, J. Mulder, C. Loveday, and C. Christopherson. 1995.
Rapid changes in human immunodeficiency virus type 1 RNA load and
appearance of drug-resistant virus populations in persons treated with lami-
vudine (3TC). J. Infect. Dis. 171:1411–1419.
154. Sharma, P. L., and C. S. Crumpacker. 1997. Attenuated replication of
human immunodeficiency virus type 1 with a didanosine-selected reverse
transcriptase mutation. J. Virol. 71:8846–8851.
155. Sharma, P. L., V. Nurpeisov, K. Lee, S. Skaggs, C. A. Di San Filippo, and
R. F. Schinazi. 2004. Replication-dependent 65R3K reversion in human
immunodeficiency virus type 1 reverse transcriptase double mutant K65R ?
L74V. Virology 321:222–234.
156. Simon, V., N. Padte, D. Murray, J. Vanderhoeven, T. Wrin, N. Parkin, M.
Di Mascio, and M. Markowitz. 2003. Infectivity and replication capacity of
drug-resistant human immunodeficiency virus type 1 variants isolated dur-
ing primary infection. J. Virol. 77:7736–7745.
157. Simon, V., J. Vanderhoeven, A. Hurley, B. Ramratnam, M. Louie, K. Dawson,
N. Parkin, D. Boden, and M. Markowitz. 2002. Evolving patterns of HIV-1
resistance to antiretroviral agents in newly infected individuals. AIDS 16:1511–
158. Sista, P. R., T. Melby, D. Davison, L. Jin, S. Mosier, M. Mink, E. L. Nelson,
R. DeMasi, N. Cammack, M. P. Salgo, T. J. Matthews, and M. L. Green-
berg. 2004. Characterization of determinants of genotypic and phenotypic
VOL. 20, 2007 CLINICAL SIGNIFICANCE OF HIV-1 REPLICATION FITNESS 577
resistance to enfuvirtide in baseline and on-treatment HIV-1 isolates. AIDS Download full-text
159. Stoddart, C. A., T. J. Liegler, F. Mammano, V. D. Linquist-Stepps, M. S.
Hayden, S. G. Deeks, R. M. Grant, F. Clavel, and J. M. McCune. 2001.
Impaired replication of protease inhibitor-resistant HIV-1 in human thy-
mus. Nat. Med. 7:712–718.
160. Sufka, S. A., G. Ferrari, V. E. Gryszowka, T. Wrin, S. A. Fiscus, G. D.
Tomaras, H. F. Staats, D. D. Patel, G. D. Sempowski, N. S. Hellmann, K. J.
Weinhold, and C. B. Hicks. 2003. Prolonged CD4?cell/virus load discor-
dance during treatment with protease inhibitor-based highly active antiret-
roviral therapy: immune response and viral control. J. Infect. Dis. 187:1027–
161. Sugiura, W., Z. Matsuda, Y. Yokomaku, K. Hertogs, B. Larder, T. Oishi, A.
Okano, T. Shiino, M. Tatsumi, M. Matsuda, H. Abumi, N. Takata, S.
Shirahata, K. Yamada, H. Yoshikura, and Y. Nagai. 2002. Interference
between D30N and L90M in selection and development of protease inhib-
itor-resistant human immunodeficiency virus type 1. Antimicrob. Agents
161a.Svarovskaia, E., J. Feng, N. Margot, F. Myrick, D. Goodman, J. Ly, K.
White, K. B. Esoda, and M. Miller. 2007. The A62V and S68G mutations
in HIV-1 reverse transcriptase partially restore the replication defect asso-
ciated with the K65R mutation, abstr. 591. Abstr. 14th Conf. Retrovir.
162. Tamiya, S., S. Mardy, M. F. Kavlick, K. Yoshimura, and H. Mistuya. 2004.
Amino acid insertions near Gag cleavage sites restore the otherwise com-
promised replication of human immunodeficiency virus type 1 variants
resistant to protease inhibitors. J. Virol. 78:12030–12040.
163. Trkola, A., H. Kuster, C. Leemann, C. Ruprecht, B. Joos, A. Telenti, B.
Hirschel, R. Weber, S. Bonhoeffer, and H. F. Gunthard. 2003. Human
immunodeficiency virus type 1 fitness is a determining factor in viral re-
bound and set point in chronic infection. J. Virol. 77:13146–13155.
164. Troyer, R. M., K. R. Collins, A. Abraha, E. Fraundorf, D. M. Moore, R. W.
Krizan, Z. Toossi, R. L. Colebunders, M. A. Jensen, J. I. Mullins, G.
Vanham, and E. J. Arts. 2005. Changes in human immunodeficiency virus
type 1 fitness and genetic diversity during disease progression. J. Virol.
165. Turner, D., B. Brenner, J. P. Routy, D. Moisi, Z. Rosberger, M. Roger, and
M. A. Wainberg. 2004. Diminished representation of HIV-1 variants con-
taining select drug resistance-conferring mutations in primary HIV-1 infec-
tion. J. Acquir. Immune Defic. Syndr. 37:1627–1631.
166. van Maarseveen, N. M., D. de Jong, C. A. Boucher, and M. Nijhuis. 2006.
An increase in viral replicative capacity drives the evolution of protease
inhibitor-resistant human immunodeficiency virus type 1 in the absence of
drugs. J. Acquir. Immune Defic. Syndr. 42:162–168.
167. van Maarseveen, N. M., A. M. Wensing, D. de Jong, M. Taconis, J. C.
Borleffs, C. A. Boucher, and M. Nijhuis. 2007. Persistence of HIV-1 variants
with multiple protease inhibitor (PI)-resistance mutations in the absence of
PI therapy can be explained by compensatory fixation. J. Infect. Dis. 195:
168. van Opijnen, T., M. C. Boerlijst, and B. Berkhout. 2006. Effects of random
mutations in the human immunodeficiency virus type 1 transcriptional
promoter on viral fitness in different host cell environments. J. Virol. 80:
169. van Opijnen, T., R. E. Jeeninga, M. C. Boerlijst, G. P. Pollakis, V. Zetterberg,
M. Salminen, and B. Berkhout. 2004. Human immunodeficiency virus type 1
subtypes have a distinct long terminal repeat that determines the replication
rate in a host-cell-specific manner. J. Virol. 78:3675–3683.
170. Verheyen, J., E. Litau, T. Sing, M. Daumer, M. Balduin, M. Oette, G.
Fatkenheuer, J. K. Rockstroh, U. Schuldenzucker, D. Hoffmann, H. Pfister,
and R. Kaiser. 2006. Compensatory mutations at the HIV cleavage sites
p7/p1 and p1/p6-gag in therapy-naive and therapy-experienced patients.
Antivir. Ther. 11:879–887.
171. Vivet-Boudou, V., J. Didierjean, C. Isel, and R. Marquet. 2006. Nucleoside
and nucleotide inhibitors of HIV-1 replication. Cell. Mol. Life Sci. 63:163–
172. Voronin, Y., J. Overbaugh, and M. Emerman. 2005. Simian immunodefi-
ciency virus variants that differ in pathogenicity differ in fitness under rapid
cell turnover conditions. J. Virol. 79:15091–15098.
173. Wainberg, M. A., W. C. Drosopoulos, H. Salomon, M. Hsu, G. Borkow, M.
Parniak, Z. Gu, Q. Song, J. Manne, S. Islam, G. Castriota, and V. R.
Prasad. 1996. Enhanced fidelity of 3TC-selected mutant HIV-1 reverse
transcriptase. Science 271:1282–1285.
174. Wang, J., C. Dykes, R. A. Domaoal, C. E. Koval, R. A. Bambara, and L. M.
Demeter. 2006. The HIV-1 reverse transcriptase mutants G190S and
G190A, which confer resistance to non-nucleoside reverse transcriptase
inhibitors, demonstrate reductions in RNase H activity and DNA synthesis
from tRNALys3 that correlate with reductions in replication efficiency.
174a.Wang, J., C. Dykes, and L. Demeter. 2006. The nucleoside resistance mu-
tations L74V and M41L?T215Y can each compensate for the reduction in
replication fitness conferred by the nonnucleoside (NNRTI) resistance
mutations K101E?G190S, abstr. 9. Abstr. 2006 Symp. Antivir. Drug Resist.
175. Weber, J., B. Chakraborty, J. Weberova, M. D. Miller, and M. E. Quinones-
Mateu. 2005. Diminished replicative fitness of primary human immunode-
ficiency virus type 1 isolates harboring the K65R mutation. J. Clin. Micro-
176. Weber, J., H. R. Rangel, B. Chakraborty, M. Tadele, M. A. Martinez, J.
Martinez-Picado, M. L. Marotta, M. Mirza, L. Ruiz, B. Clotet, T. Wrin,
C. J. Petropoulos, and M. E. Quinones-Mateu. 2003. A novel TaqMan
real-time PCR assay to estimate ex vivo human immunodeficiency virus
type 1 fitness in the era of multi-target (pol and env) antiretroviral therapy.
J. Gen. Virol. 84:2217–2228.
177. Wei, X., S. K. Ghosh, M. E. Taylor, V. A. Johnson, E. A. Emini, P. Deutsch,
J. D. Lifson, S. Bonhoeffer, M. A. Nowak, B. H. Hahn, et al. 1995. Viral
dynamics in human immunodeficiency virus type 1 infection. Nature 373:
178. Weinstock, H. S., I. Zaidi, W. Heneine, D. Bennett, J. G. Garcia-Lerma,
J. M. Douglas, Jr., M. LaLota, G. Dickinson, S. Schwarcz, L. Torian, D.
Wendell, S. Paul, G. A. Goza, J. Ruiz, B. Boyett, and J. E. Kaplan. 2004.
The epidemiology of antiretroviral drug resistance among drug-naive HIV-
1-infected persons in 10 US cities. J. Infect. Dis. 189:2174–2180.
179. White, K. L., N. A. Margot, T. Wrin, C. J. Petropoulos, M. D. Miller, and
L. K. Naeger. 2002. Molecular mechanisms of resistance to human immu-
nodeficiency virus type 1 with reverse transcriptase mutations K65R and
K65R?M184V and their effects on enzyme function and viral replication
capacity. Antimicrob. Agents Chemother. 46:3437–3446.
180. Wirden, M., I. Malet, A. Derache, A. G. Marcelin, B. Roquebert, A. Simon,
M. Kirstetter, L. M. Joubert, C. Katlama, and V. Calvez. 2005. Clonal
analyses of HIV quasispecies in patients harboring plasma genotype with
K65R mutation associated with thymidine analogue mutations or L74V
substitution. AIDS 19:630–632.
181. Wu, H., Y. Huang, C. Dykes, D. Liu, J. Ma, A. S. Perelson, and L. M.
Demeter. 2006. Modeling and estimation of replication fitness of human
immunodeficiency virus type 1 in vitro experiments by using a growth
competition assay. J. Virol. 80:2380–2389.
181a.Yeh, F. L., L. Miranda, and D. Kuritzkes. 2006. The effect of the M184V
substitution in HIV-1 reverse transcriptase on DNA 3?-end and RNA
5?-end directed RNase H activity, abstr. 600. Abstr. 13th Conf. Retrovir.
182. Yerly, S., S. Jost, A. Telenti, M. Flepp, L. Kaiser, J. P. Chave, P. Vernazza, M.
Battegay, H. Furrer, B. Chanzy, P. Burgisser, M. Rickenbach, M. Gebhardt,
M. C. Bernard, T. Perneger, B. Hirschel, and L. Perrin. 2004. Infrequent
transmission of HIV-1 drug-resistant variants. Antivir. Ther. 9:375–384.
183. Yerly, S., S. Vora, P. Rizzardi, J. P. Chave, P. L. Vernazza, M. Flepp, A.
Telenti, M. Battegay, A. L. Veuthey, J. P. Bru, M. Rickenbach, B. Hirschel,
L. Perrin, and the Swiss HIV Cohort Study. 2001. Acute HIV infection:
impact on the spread of HIV and transmission of drug resistance. AIDS
184. Zhang, H., Y. Zhou, C. Alcock, T. Kiefer, D. Monie, J. Siliciano, Q. Li, P.
Pham, J. Cofrancesco, D. Persaud, and R. F. Siliciano. 2004. Novel single-
cell-level phenotypic assay for residual drug susceptibility and reduced
replication capacity of drug-resistant human immunodeficiency virus type 1.
J. Virol. 78:1718–1729.
185. Ziermann, R., K. Limoli, K. Das, E. Arnold, C. J. Petropoulos, and N. T.
Parkin. 2000. A mutation in human immunodeficiency virus type 1 pro-
tease, N88S, that causes in vitro hypersensitivity to amprenavir. J. Virol.
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