JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2010, p. 4035–4043
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 11
Human Immunodeficiency Virus Type 1 Protease Inhibitor
Drug-Resistant Mutants Give Discordant Results
When Compared in Single-Cycle and
Multiple-Cycle Fitness Assays?
Carrie Dykes,1* Hulin Wu,2Matthew Sims,1† Jeanne Holden-Wiltse,2and Lisa M. Demeter1
Department of Medicine1and Department of Biostatistics,2University of Rochester School of
Medicine and Dentistry, Rochester, New York
Received 22 March 2010/Returned for modification 11 June 2010/Accepted 26 August 2010
The replication fitness of HIV-1 drug-resistant mutants has been measured using either multiple-cycle or
single-cycle assays (MCAs or SCAs); these assays have not been systematically compared. We developed an
MCA and an SCA that utilized either intact or env-deleted recombinant viral vectors, respectively, in which
virus-infected cells were detected by flow cytometry of a reporter gene product. Fitness was measured using
each assay for 11 protease mutants, 9 reverse transcriptase mutants, and two mutants with mutations in gag
p6, which is important for the release of virus particles from the cell membrane. In the SCA, fitness (replication
capacity [RC]) was defined as the proportion of cells infected by the mutant compared to the wild type 40 h
after infection. MCA fitness (1?s) was determined by comparing the changes in the relative proportions of cells
infected by the mutant and the wild type between 3 and 5 days after infection. Five protease mutants showed
statistically different fitness values by the MCA versus the SCA: the D30N, G48V, I50V, I54L, and I54M
mutants. When all the mutants were ranked in order from most to least fit for both assays, 4 protease mutants
moved more than 5 positions in rank: the D30N, I54L, I54M, and V82A mutants. There were no significant
differences in fitness for the gag p6 or reverse transcriptase mutants. We propose that discordant results in the
MCA and SCA are due to alterations in late events in the virus life cycle that are not captured in an SCA, such
as burst size, cell-to-cell transmission, or infected-cell life span.
Fitness, as defined in population biology, is the ability of a
variant to contribute to successive generations (reviewed in
reference 16). The more fit a variant, the more likely it is to
contribute its genotype to its offspring. Human immunodefi-
ciency virus type 1 (HIV-1) occurs as a quasispecies in patients,
in which many genetically related but distinctly different vari-
ants exist together as an evolving group (17). It has been
hypothesized that the most dominant variant in a patient is that
which is most fit under current selective pressures (11). When
selective pressures change, for example, by the introduction of
drug therapy or through action by the immune system, the
most dominant variant will change. There are phenomena in
patients in which fitness might play a role, such as the likeli-
hood that a variant will be transmitted and how quickly the
The role of fitness in the development of drug resistance,
transmission, and disease progression has been studied using
many types of fitness assays. These studies have found that
drug-resistant variants replicate less efficiently than their drug-
sensitive counterparts (4, 13, 15, 24, 32, 36, 39, 48) and that
fitness is one factor that determines the relative prevalence of
drug-resistant variants in a patient failing drug therapy. Studies
of the role of fitness in disease progression and transmission
have been performed using both single-cycle and multiple-
cycle assays (SCAs and MCAs) and have yielded conflicting
results (42). Therefore, it is still unclear what role fitness may
play in pathogenesis and what impact the assay itself has on the
In order to understand the role of fitness in HIV-1 patho-
genesis, more information is needed on how well different cell
culture fitness assays correlate. Five different characteristics
have been used to describe such fitness assays: whole virus
versus recombinant virus, growth competitions versus parallel
infections, direct measurement of a viral gene product versus a
reporter gene, primary cells versus a cell line, and a single cycle
versus multiple cycles (19). MCAs are performed with whole
virus or recombinant vectors that have all the genes in the
genome that are essential for growth in culture and can un-
dergo multiple rounds of replication over the course of the
experiment (5, 21, 27, 31). SCAs are performed with a recom-
binant vector in which in an essential gene, usually envelope, is
deleted and which can undergo only a single round of replica-
tion (51). SCAs have the advantage of being performed in a
shorter time frame, usually 48 h, and therefore have a higher
throughput, than MCAs, which take days to weeks to com-
plete. However, it is not known whether mutants of HIV-1
have the same relative fitness when compared against the wild
type (WT) in a multiple-cycle assay and in a single-cycle assay.
We hypothesized that the SCA would detect abnormalities in
virus entry, reverse transcription, integration, and protein ex-
pression but would not reflect abnormalities in late stages of
* Corresponding author. Mailing address: University of Rochester
School of Medicine and Dentistry, Infectious Diseases Division, 601
Elmwood Avenue, Box 689, Rochester, NY 14642. Phone: (585) 273-
4104. Fax: (585) 442-9328. E-mail: email@example.com.
† Present address: Department of Infectious Diseases, William
Beaumont Hospital, 3601 W. Thirteen Mile Rd., Royal Oak, MI 48073.
?Published ahead of print on 8 September 2010.
the virus replication cycle, such as burst size, cell-to-cell trans-
mission, or infected-cell life span. In contrast, an MCA should
reflect all the steps in the virus life cycle. In addition, small
differences in fitness may be amplified over several rounds of
replication, resulting in a larger fitness deficit in the MCA than
in the SCA.
In order to determine whether the use of an MCA versus an
SCA results in different relative fitness values, we developed an
SCA analogous to a flow cytometry-based MCA that has been
described previously (21) and compared the two assays using a
panel of HIV-1 mutants resistant to protease and reverse
transcriptase (RT) inhibitors.
MATERIALS AND METHODS
Reagents. The following reagents were obtained through the AIDS Research
and Reference Reagent Program, Division of AIDS, National Institute of Al-
lergy and Infectious Diseases: SV-A-MLV-env, from Nathaniel Landau and Dan
Littman (29); the PM1 cell line, a clonal derivative of HUT 78 that is permissive
for both macrophage-tropic and lymphocyte-tropic strains of HIV-1, from Mar-
vin Reitz (33); and the MT-4 T cell line, from Douglas Richman (26, 30). The 293
and COS-1 cell lines were obtained from the American Type Culture Collection
(ATCC, Rockville, MD). Plasmid pHCMV-g expresses the vesicular stomatitis
virus g protein (VSV-g) under the control of the human cytomegalovirus pro-
moter (18, 50). Fetal bovine serum (FBS) was obtained from Valley Biomedical
(Winchester, VA). Restriction enzymes were obtained from either New England
Biolabs (Beverly, MA) or MBI Fermentas (Hanover, MD). Fluorescein isothio-
cyanate (FITC)-conjugated-mouse anti-rat Thy1.1 (HIS51) and R-phycoerythrin
(PE)-conjugated rat anti-mouse Thy1.2 (30-H12) monoclonal antibodies were
obtained from BD Biosciences (San Jose, CA).
Cell culture. PM1 and MT-4 cells were cultured in the presence of RPMI
medium (Cellgro, Herndon, VA) supplemented with 10% FBS, L-glutamine (2
mM), penicillin (100 U/ml), and streptomycin (100 U/ml). 293 and COS-1 cells
were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% FBS,
penicillin (100 U/ml), and streptomycin (100 U/ml). Primary human peripheral
blood mononuclear cells (PBMCs) isolated from HIV-negative donors were
purified by Ficoll-Hypaque density centrifugation, stimulated for 2 days with 5
?g/ml phytohemagglutinin P (PHA-P; Sigma-Aldrich) and 5% interleukin-2 (IL-
2), and cultured in RPMI 1640 supplemented with 20% (vol/vol) FBS, penicillin
(100 U/ml), streptomycin (100 U/ml), and 5% IL-2. All cells were propagated
under 5% CO2at 37°C.
Construction of the pDAT1 and pDAT2 HIV-1 vectors. The construction of the
pAT1 and pAT2 vectors has been described previously (21). Briefly, the pAT1
and pAT2 constructs are identical to NL4-3 except that they contain the mouse
Thy1.1 or Thy1.2 gene, respectively, in place of nef, have silent XmaI and XbaI
restriction sites that flank RT to facilitate the cloning of patient RT sequences,
and have the XbaI site removed from between the 3? long terminal repeat (LTR)
and the pUC19 plasmid sequence. pDHIV3.Thy1.1 is derived from pNL4-3 and
has a 700-bp deletion in env and the mouse Thy1.1 gene cloned in place of nef
(18). env-deleted versions of pAT1 and pAT2 were made by replacing the
pDHIV.3.Thy1.1 to produce pDAT1 and pDAT2, respectively (Fig. 1).
Introduction of drug resistance mutations. Mutations in protease and RT
were introduced into pRHAXX (20) using the QuikChange II mutagenesis kit
(Stratagene). pRHAXX was made previously by introducing silent XmaI and
XbaI restriction sites into pRHA, which was created by subcloning the pol gene
of pNL4-3 into the phagemid vector pTZ18U (Bio-Rad, Hercules, CA) by using
SphI and EcoRI restriction endonuclease sites (20, 24). The generation of pAT2
containing either the L90M, D30N, K103N, V106A, G190S, or P236L mutant has
been described previously (21). Other mutants used in this study were generated
using pRHAXX and the primers given in parentheses (the sense version of the
primer set is shown) as follows: the protease L33F (5?-GGAGCAGATGATAC
AGTATTCGAAGAAATGAATTTGCCA), G48V (5?-AAACCAAAAATGAT
AGTGGGAATTGGAGGTTTT), I50L (5?-AAAAATGATAGGGGGACTTG
GAGGTTTTATCAAA), I50V (5?-CCAAAAATGATAGGGGGAGTTGGAG
GTTTTATCAAAGTA), I54L (5?-GGGGGAATTGGAGGTTTTCTCAAAGT
AAGACAGTATGAT), I54M (5?-GGGGGAATTGGAGGTTTTATGAAAGT
AAGACAGTATGAT), V82A (5?-GTAGGACCTACACCTGCCAACATAAT
TGGAAGAAAT), V82T (5?-TTAGTAGGACCTACACCTACCAACATAAT
TGGAAGAAATCTG), and I84V (5?-CTACACCTGTCAACATAATTGGAA
GAAATCTG) mutants; the RT K65R (5?-GTATTTGCCATAAAGAGAAAA
GACAGTACTAAATGG), K70R (5?-GAAAAAAGACAGTACTAGATGGA
GAAAATTAGTAG), Q151L (5?-CCACTGGGATGGAAAGGATCACCAGC
AATATTCC) (used to generate the template for Q151M), Q151M (5?-CCAA
TGGGATGGAAAGGATCACCAGCAATATTCC), M184V (5?-CATCTATC
AATACGTGGATGATTTGTATGTAGG), and T215Y (5?-TGAGGTGGGG
ATTTTACACACCAGACAAAAAAC) mutants; and the Gag p6 PTAPAPP
The antisense primers used for mutagenesis were the reverse and complement
of each sense primer. The resultant clones were sequenced to confirm the
presence of the mutation and the absence of spurious mutations. Protease in-
hibitor resistance mutations and p6 proline-threonine-alanine-proline (PTAP)
insertion mutations were introduced into pAT2 and pDAT2 by subcloning the
ApaI-XmaI fragment from the mutant pRHAXX constructs, and RT mutations
were introduced by subcloning the XmaI-AgeI fragment from the mutant
pRHAXX constructs. All mutant pDAT2 and pAT2 clones were sequenced to
verify the presence of the mutation and the integrity of the cloning sites.
Generation of virus stocks. pAT1WT and pAT2 mutant virus stocks were
made by transfecting 293 cells as described previously (21). Five micrograms of
pDAT1WT or pDAT2 mutant constructs was used to transiently transfect 1 ?
106COS-1 or 4 ? 106293 cells seeded in 10-cm-diameter plates the day before,
along with 5 ?g of pHCMV-g (expressing the VSV-g envelope) or pSV-A-MLV-
ENV (expressing the murine leukemia virus [MLV] envelope), by using 25 ?l of
Lipofectamine LTX (Invitrogen). DNA and Lipofectamine LTX were incubated
for 30 min in 2 ml of Opti-MEM (Invitrogen). Eight milliliters of DMEM
containing 10% FBS and Pen/Strep were added to each transfection mixture
withthe same fragmentfrom
FIG. 1. HIV-1 vectors pDAT1, used for the SCA (A), and pAT1, used for the MCA (B). The open reading frames for gag, pol, the accessory
genes, Thy1 markers, and long terminal repeats are shown, as are the bacterial pUC19 sequences (pUC) and the 5? cellular flanking sequence.
Vertical lines indicate the locations of restriction enzymes. A black triangle indicates the location of the deletion in the envelope in the pDAT
vector used for the SCA.
4036DYKES ET AL.J. CLIN. MICROBIOL.
before it was added to the COS-1 cells. Supernatants were harvested 72 h later
and were clarified by centrifugation at 400 ? g. HIV-1 capsid protein (p24)
quantitation was performed on virus stocks using an HIV-1 p24 enzyme-linked
immunosorbent assay (ELISA) (Perkin-Elmer, Norwalk, CT), and 25 to 100 ng
of each virus stock was used per infection. Virus stocks were tested in triplicate
on 96-well plates, and competing stocks were tested in the same assay.
VSV-g protein quantitation. VSV-g protein quantitation was performed by
ELISA using a horseradish peroxidase-conjugated anti-VSV-g antibody (Abcam,
Cambridge, MA). DAT virus stocks were tested in triplicate on 96-well plates,
and competing stocks were tested on the same assay. Virus stocks were diluted
1:2 with phosphate-buffered saline (PBS), and 200 ?l was incubated with 20 ?l of
5% Triton X-100 and 0.02% sodium azide in PBS. Stocks were incubated for 2 h
at 37°C and were then washed twice with wash solution (0.2% Tween 20 in PBS).
Blocking solution (2% bovine serum albumin [BSA] and 0.2% Tween 20 in PBS)
was then added, and the mixture was incubated for 90 min at room temperature.
The plates were washed twice, and 100 ?l of anti-VSV-g antibody diluted 1:500
in blocking solution was added for 2 h at room temperature. The plates were
washed 4 times, and 100 ?l of tetramethylbenzidine(TMB) substrate (Pierce) was
incubated for 1 h at room temperature in the dark. The reaction was stopped by
the addition of 100 ?l of 2 M sulfuric acid, and absorbance was measured at 450
nm. A single wild-type virus stock was used as a control for each assay. The ratio
of the optical density (OD) of the test stock to that of the control stock was used
to compare the input inoculum for the SCA to the inoculum calculated using the
p24 content and was used to determine the input inoculum relative to wild-type
virus in some SCAs.
Multiple-cycle and single-cycle growth competition assays. The MCAs in PM1
cells were performed as previously described (21). Briefly, 7 million PM1 cells
were coinfected with equal amounts of wild-type and mutant viruses as deter-
mined by p24 capsid protein quantitation. At days 3, 4, 5, and 6, half the culture
was removed and replaced with fresh medium. The number of viable cells was
determined by trypan blue staining. One-half million cells were stained with the
anti-Thy1.1 or anti-Thy1.2 antibody alone, or with the anti-Thy1.1 and anti-
Thy1.2 antibodies together, diluted 1:100 and 1:200, respectively, in fluores-
cence-activated cell sorter (FACS) buffer (PBS with 0.02% FBS, 0.02% sodium
azide, and 0.5 mM EDTA). The MCA performed in other cell types was the
same as that in PM1 cells except for the virus inoculum and cell number (5 ng p24
of each virus infected 14 million MT4 cells subcultured at 200,000 cells/ml, and
70 ng p24 of each virus infected 10 million human PBMCs, which were subcul-
tured at 2 million cells/ml). These inocula were determined empirically so as to
give approximately 0.5% infected cells 3 days after infection.
For the SCA, 1 million PM1 cells were coinfected with 100 ng p24 (or, in some
cases, 1.0 OD unit of VSV-g) each of WT and mutant virus. Cells were incubated
with virus at 37°C in a total volume of 0.8 ml for 1 h; then they were washed with
PBS, centrifuged at 500 ? g, and seeded at 200,000 cells/ml in the appropriate
medium. At 40 h, trypan blue staining was used to determine the viable cell
count. At this time point, 0.5 million cells were also stained with the anti-Thy1.1
and anti-Thy1.2 antibodies and were analyzed by FACS using the same methods
as those for the MCA (21).
Fitness calculation and statistical analysis. For the MCA, the 1?s value was
determined using the method of Wu et al. (49). A fitness calculator, available at
http://bis.urmc.rochester.edu/vFitness/, was used to obtain the average 1?s value
by using the numbers of infected cells from days 3 to 5. For the SCA, fitness was
calculated using the following equation: replication capacity (RC) ? (% mutant-
infected cells)/(% WT-infected cells). Cells that were infected with both mutant
and WT viruses were included in both the numerator and the denominator. For
assay and stock variability studies, the absolute deviation of each observation
from the sample median (robust measure of variability) was constructed. Two-
way analysis of variance (ANOVA) was performed to test for variability differ-
ences in mean deviations between stock preparations. Two-way ANOVA tests
were also performed to account for any differences between mutants in testing
for relative fitness value differences by assay type or cell line. Significant effects
were followed with Wilcoxon two-sample exact tests due to the small sample
sizes. All reported P values are Bonferroni adjusted for multiple comparisons
within each experiment, maintaining an overall experiment-wide alpha value of
0.05. Log transformations were taken to normalize relative fitness value (RFV)
measurements for ANOVA tests. Analyses were performed using SAS, version
9.2 (SAS Institute, Cary, NC).
Assay design. We designed a single-cycle fitness assay that
can differentiate wild-type from mutant virus in the same cul-
ture and that is otherwise the same as a previously described
MCA (21). Both the wild-type MCA and SCA vectors have the
mouse Thy1.1 gene cloned in place of nef and are identical to
each other except that the SCA vector pDAT has a 570-bp
deletion in the env gene (Fig. 1). Virus derived from the pDAT
vectors was pseudotyped with either VSV-g or MLV env. Site-
directed mutants were cloned into the Thy1.2 versions of the
pAT and pDAT constructs, and independent virus stocks were
produced by transfection. Cells were coinfected with wild-type
and mutant viruses, and the proportion of cells infected with
wild-type or mutant virus was determined by flow cytometry
(Fig. 2). Each culture was stained with each Thy antibody
individually; one aliquot was also stained with both antibodies.
Thresholds for Thy1.1 and Thy1.2 positivity were determined
by using the heterologous anti-Thy antibody as a control on the
singly stained samples; one blinded observer set all gates. We
compared the replication of mutant and wild-type reference
viruses using the dually stained sample and compared the RC
value for each mutant as determined by the SCA with the 1?s
value determined by the MCA.
Assay variability. We have previously observed that for the
MCA there is more variability between cultures than there is
between virus stocks (data not shown). Since SCA stocks are
made by cotransfection of an envelope vector and the parental
FIG. 2. Experimental design of the SCA. Circular versions of the
constructs from Fig. 1 are shown with the wild type in black and the
mutant in gray. COS-1 or 293 cells were cotransfected with pHCMV-g
or pSV-A-MLV-ENV and either wild-type pDAT1 (pDAT1WT) or
mutant pDAT2 (pDAT2MUT). Wild-type (black) and mutant (gray)
virus stocks are represented by hexagons. The titers of virus stocks
were determined by measuring the p24 or VSV-g protein content. PM1
cells were coinfected with equal amounts of wild-type and mutant
viruses. After 40 h, cells were harvested, and a subset of cells was
stained with either an anti-Thy1.1 antibody, an anti-Thy1.2 antibody,
or both. Four different types of cells can be observed during analysis:
uninfected cells, cells infected with wild-type virus only, cells infected
with mutant virus only, and cells infected with both viruses (dually
VOL. 48, 2010HIV FITNESS IN SINGLE- AND MULTIPLE-CYCLE ASSAYS 4037
vector, we wanted to determine if the assay-to-assay variability
was higher than the stock-to-stock variability. The assay-to-
assay variability of the SCA was determined by measuring the
fitness of a VSV-g-pseudotyped stock derived from a single
transfection in six independent cultures set up on six different
days. Each culture contained a wild-type reference derived
from a single transfection. The stock-to-stock variability using
VSV-g- or MLV-pseudotyped virus was determined by mea-
suring the RCs of nine pseudotyped stocks (three independent
stocks made on each of three different days). Each of these
infections contained a wild-type reference derived from a sin-
gle transfection. The G190S mutant, a reverse transcriptase
mutant known to have reduced fitness in this MCA, and the
K103N mutant, a reverse transcriptase mutant with preserved
fitness in this MCA (21), were used for this comparison. We
compared the mean absolute deviations (measure of variabil-
ity) of the RCs generated from the VSV-g assay-to-assay vari-
ability tests with those of the RCs generated from the VSV-g
stock-to-stock variability tests. Overall, there was significantly
less assay variability (P, 0.0437; two-way ANOVA main effect)
than stock variability. This comparison was the same for the
G190S and K103N mutants (P, 0.1038).
Because MLV and VSV-g envelope proteins are commonly
used to pseudotype HIV-1 (2, 3, 42, 43, 45), we compared the
variability in RC values generated by viruses pseudotyped with
these two envelopes and also compared the RC values to the
1?s values derived from the MCA. The stock-to-stock vari-
ability of the MCA was evaluated with four stocks, each made
on a different day (Fig. 3). The mean RC for the K103N
mutant was not significantly different when either MLV or
VSV-g was used as the envelope (P, 0.4961). The mean RC for
the G190S mutant was somewhat higher when MLV was used
as an envelope (0.63 versus 0.38; P, 0.01); however, this did not
hold true for the other mutants tested (the D30N, L90M, and
V106A mutants [data not shown]). In order to determine if
there was more variability between the SCA and the MCA, we
compared the mean absolute deviations of the SCA MLV and
SCA VSV-g data for the K103N or G190S mutant with the
MCA data for the K103N or G190S mutant. Overall, there was
a marginally significant (P, 0.0501) difference in the variabil-
ity (mean deviations) between stock preparations. Further
posthoc comparison tests showed no significant differences in
variability between any of the stock preparations (P, 0.9363)
for the G190S mutant. For the K103N mutant, there was a
significant difference in mean variability (P, 0.0405) when all 3
stock preparations were compared; however, any pairwise
comparison of stock preparations was statistically nonsignifi-
cant (P, ?0.05). These results suggest, particularly for the
K103N mutant, more variability in the VSV-g stocks than in
the VSV-g assays and more variability in the VSV-g SCA than
in the MLV SCA. Therefore, we tested 6 independent stocks in
single competitions for the remaining SCA studies and chose
the MLV envelope for the remaining assays.
Effect of the virus input measurement method on RC. One
potential explanation for the greater variability in the SCA
than in the MCA is that the SCA only measures the replication
of the mutant versus the wild-type reference strain at a single
time point. Variation in the measurement of the relative inoc-
ula of wild-type and mutant viruses would therefore have a
greater impact on the RC value than on the (1?s) value de-
rived from the MCA, which is based on the relative prevalence
of mutant versus wild-type virus using three time points. Since
virus was pseudotyped by cotransfecting the DAT construct
with the MLV envelope or VSV-g expression construct, we
hypothesized that there could be different efficiencies of enve-
lope incorporation into the different stocks, which could im-
pact relative infectivity, and therefore RC. To test this, we
measured the VSV-g protein content of each K103N and
G190S stock by ELISA, measured the RC by coinfecting cells
with equal amounts of VSV-g protein, and compared that to
the RC obtained when the p24 protein content was used. Since
the assay variability for the SCA was shown to be low, we
tested each stock only once. There were no significant differ-
ences in the mean RC values for the K103N mutant (RC, 1.47
for p24-defined input versus 1.64 for VSV-g; P, 0.7422). There
were also no differences in RC for the G190S mutant depend-
ing on whether p24 or VSV-g was used to determine input
(0.38 for p24 versus 0.39 for VSV-g; P, 0.8125). These findings
indicate that similar ratios of VSV-g to p24 antigen exist in the
different stocks and that this explanation does not account
for the greater variability in RC observed with VSV-g-
Comparing the SCA to the MCA. We next determined if the
relative fitness values for different drug-resistant mutants
would be similar by the SCA and the MCA. We chose to test
the mutants in the SCA using MLV envelope, since this assay
appeared to be less variable than the assay that utilized VSV-
g-pseudotyped stocks. The mean fitness values of the mutants
tested in the MCA ranged widely, from 1.36 to 0.18 (Fig. 4A).
When the same mutants were tested in the MLV-SCA, the
FIG. 3. Assay and stock variability. For assay variability, single
VSV-g/DAT2G190S and VSV-g/DAT2K103N stocks were tested
against a single WT stock in 6 independent assays set up on different
days. For stock variability, nine independently transfected G190S or
K103N stocks were tested against a single WT stock by the SCA. Four
independently transfected G190S or K103N stocks were tested on
different days, each in triplicate, against the same WT stock for the
multiple-cycle assays. The relative input used for each stock was de-
termined by the p24 protein content. The relative fitness value for
either the SCA (RC) or the MCA (1?s) is shown along the y axis. Each
box indicates the upper and lower quartiles; the sold line within the box
represents the mean; and the whiskers show the range.
4038DYKES ET AL.J. CLIN. MICROBIOL.
mean RC values had a range of 4.26 to 0.046 (Fig. 4B). When
we conotrolled for the effect of the mutant (P, ?0.0001), there
was no significant difference in mean RFV between the MCA
and the SCA (P, 0.10), although the effect of the assay is
modified by the significant interaction of mutant and assay
Based on the model of all 22 mutants, further multiple
comparison testing of each mutant showed that the relative
fitness value for each mutant did not differ by assay, with the
exception of 3 mutants in the MCA and 2 mutants in the SCA
replicating more poorly than in the alternative assay (Table 1).
The D30N mutant, whose relative fitness was only slightly
reduced in the SCA compared to that of the wild type, signif-
icantly replicated more poorly in the MCA. Similarly, the
G48V and I54L mutants had reduced relative fitness in the
MCA compared to the relative fitness in the SCA. In contrast,
the I50V and I54M mutants had reduced relative fitness in the
SCA compared to the relative fitness in the MCA.
FIG. 4. Comparison of the SCA with the MCA. (a) MCA. The site-directed mutants tested against the WT are shown along the x axis. The 1?s
value, as calculated from the fitness website, is shown along the y axis. Each bar represents the average and standard deviation for at least 6
replicate cultures. (b) SCA. The relative input used for each stock was determined by the p24 protein content. The site-directed mutants tested
against the WT are shown along the x axis. The RC value is shown along the y axis. Each bar represents the average and standard deviation for
at least 6 independent virus stocks tested in single cultures and against the same WT stock. Circles highlight the positions of the D30N and I54L
mutants, and squares highlight those of the I54M and V82A mutants, on each x axis.
VOL. 48, 2010 HIV FITNESS IN SINGLE- AND MULTIPLE-CYCLE ASSAYS4039
We hypothesize that the MCA and SCA fitness values take
into account different steps of the virus life cycle. If this is true,
statistical comparison of the absolute fitness values of the mu-
tant tested in each assay may not be valid. We think it more
biologically relevant to compare the rank order among mutants
between the two assays. This analysis would help determine
how much a mutant’s fitness changes relative to that of the wild
type. Therefore, we ranked the mutants from most fit to least
fit in the MCA and determined if any mutants changed more
than 5 rank positions in the SCA. When this analysis was
performed, the D30N and I54L mutants (Fig. 4; marked with
circles) moved 8 and 6 spots, respectively, and the I54M and
V82A mutants (Fig. 4; marked with boxes) both moved down
11 spots. The D30N and I54L mutants were more fit in the SCA,
and the I54M and V82A mutants were less fit in the SCA.
Effect of cell line on MCA. Cong et al. have also tested the
relative fitness of several mutants resistant to nucleoside RT
inhibitors and nonnucleoside RT inhibitors (NNRTIs) by using
an MCA (13). Of note is that some nucleoside-resistant mu-
tants that showed reduced fitness in their MCA did not have a
discernible replication deficit in our MCA. Two important
differences between their assay and ours were the cells used
(PM1 cells in our assay versus MT-4 cells and PBMCs in theirs)
and the viral sequence backbone (NL4-3 versus HXB-2). We
therefore evaluated whether the cell line used in the assay
could affect the relative fitness of a subset of mutants, using
MT-4 cells or primary human PBMCs (Fig. 5). The overall test
of the main effect of cell line was not significant (P, 0.8055),
although there was a significant interaction between the mu-
tant and the cell line (P, ?0.0001). However, further compar-
ison testing showed that the relative fitness of each of these
mutants in the PM1 cell line did not differ significantly
(P, ?0.05) from the relative fitness based on either the MT4 or
the PBMC line.
We have developed two HIV-1 replication fitness assays that
use flow cytometry to detect the number of cells infected by
a drug-resistant mutant, compared to the number infected by a
WT reference strain in the same culture. The SCA utilizes a
vector in which env is deleted and viruses are pseudotyped with
MLV envelope. Therefore, viruses undergo a single round of
infection with no subsequent rounds. Mutant virus produced
after transfection is normalized for p24 capsid protein against
WT, and cells are subsequently infected with equal amounts of
WT and mutant viruses. The SCA vector has the mouse Thy
gene cloned in place of nef, resulting in the expression of Thy
protein on the surfaces of infected cells. In contrast, the MCA,
which we have described previously (21), utilizes Thy-express-
ing virus with an intact envelope; viruses therefore undergo
TABLE 1. Comparison of the relative fitnesses of protease mutants
with different relative fitnesses in the single-cycle assay
versus the multiple-cycle assaya
Result (95% CI) by:
SCA (RC)MCA (1?s)
0.80 ? 0.16 (0.68–0.92)
0.34 ? 0.13 (0.21–0.47)
0.89 ? 0.16 (0.72–1.06)
0.036 ? 0.006 (0.030–0.043) 0.29 ? 0.045 (0.24–0.33) 0.0484
0.34 ? 0.09 (0.25–0.43) 0.79 ? 0.04 (0.75–0.83)
0.32 ? 0.10 (0.21–0.42)
0.18 ? 0.02 (0.16–0.20)
0.49 ? 0.11 (0.38–0.60)
aSCA, single-cycle assay; RC, replication capacity; CI, confidence interval;
MCA, multiple-cycle assay.
FIG. 5. Measurement of fitness by the MCA using different cell lines. The relative input used for each stock was determined by the p24 protein
content. The site-directed mutants tested against the WT are shown along the x axis. The 1?s value, as calculated from the fitness website, is shown
along the y axis. Each bar represents the average and standard deviation for at least 6 replicate cultures. Mutants were tested against the WT in
PM1 cells (filled bars), MT4 cells (shaded bars), and human PBMCs (open bars).
4040DYKES ET AL. J. CLIN. MICROBIOL.
several rounds of replication during the course of the experi-
We used these flow cytometry-based MCAs and SCAs to test
a panel of 22 drug-resistant mutants. The most striking finding
of this study was that four protease inhibitor-resistant mutants
had marked differences in relative fitness by the two assays
(Fig. 4). The replication deficit of the D30N mutant was mod-
erate (20% reduction relative to WT) in the SCA and more
pronounced (68% reduction) in the MCA (Table 1). Similarly,
the I54L mutant had a fitness similar to that of the wild type in
the SCA but a significant reduction in the MCA (Table 1). In
contrast, the I54M and V82A mutants had greater decreases
in fitness in the SCA, 66% and 56%, respectively, and were
similar to the WT in the MCA (Fig. 4). The G48V and I50V
mutants also had significant differences in their fitness values
between the two assays; however, their relative fitness com-
pared to that of the wild type in both assays was very poor
We hypothesize that the SCA, in which the lack of an en-
velope gene results in noninfectious progeny, should detect
abnormalities in virus entry, reverse transcription, integration,
and protein expression but would be insensitive to changes in
the virus life cycle after early gene expression, such as the
production of infectious virions from infected cells (i.e., burst
size), the efficiency of cell-to-cell transmission, or the life span
of infected cells. The MCA should reflect all these steps in the
virus life cycle and may also reflect compensatory interactions
between events that require multiple rounds of infection and
those that can be measured by both assays (e.g., increases in
cell-to-cell transmission could completely or partially compen-
sate for reductions in protease activity). We believe that dis-
cordant results for the protease mutants provide important
mechanistic clues as to which specific steps in the HIV repli-
cation cycle are affected by the mutation. The protease activity
of the D30N, I54L, I54M, and V82A mutants correlates with
the relative fitness measured in our SCA, indicating that in-
fectivity is one step of the viral life cycle measured by the SCA
(1, 10, 34). The fitness defects of the D30N and I54L mutants
were even more pronounced in the MCA, which could be due
to defects in both maturation and steps in virus spread that
occur only in the MCA. One possible explanation as to why the
I54M and V82A mutants are more fit in the MCA is that
defects in particle production and maturation, which would be
detected in both assays, are compensated for by improvements
in steps of viral replication that are measured only in the MCA.
This would result in an overall improvement in fitness in the
MCA for the V82A mutant.
To determine whether burst size might explain the fitness
differences seen for the protease mutants, two duplication mu-
tants of the PTAP motif of p6 were made. This motif has been
shown to help the release of particles from the cell by seques-
tering TSG101 (22, 25). The duplications used in this study
have been observed in drug-experienced patients and in cyto-
toxic T-lymphocyte (CTL) escape variants (9, 41). It is hypoth-
esized that these duplications increase the virus’s ability to
release particles from the cell surface. In our assays, the fitness
of these mutants was not significantly different between the two
assays. Therefore, either these mutants do not increase particle
production, or burst size is measured in both assays and does
not influence only the MCA fitness value.
The SCA has two technical differences from the MCA that
could cause the potential variability that was observed in this
study. The first is the need to cotransfect producer cell lines
with the vector and envelope. Differences in the abilities of the
two clones to be comparatively expressed from one transfec-
tion to another can introduce variability in the amount of
envelope protein that is incorporated into the virion. Even in
the face of equal expression, there is also the potential for
variability in the amount of envelope protein that is function-
ally incorporated into virions. A second difference is that the
RC is determined by a measurement at a single time point.
Since relative fitness is not measured as a change over time, as
in the MCA, the assay is much more sensitive to the relative
amounts of WT and mutant viruses that are added. These two
differences potentially create more variability in the RC values
of the SCA than in the MCA values and result in the need to
average the results for several different stocks.
For the panel of mutants we tested, some previously pub-
lished studies gave results similar to ours and some gave dis-
similar results. There is no previously published information
comparing the fitness of HIV-1 drug-resistant mutants in mul-
tiple- versus single-cycle assays. In one previous report, the
fitness deficit of the D30N mutant was 40% (40) compared to
the WT in an SCA and was reduced 60% in an independent
MCA (36). Like our studies, this MCA study also showed that
the fitness of the L90M mutant was similar to that of the WT
and greater than that of the D30N mutant. Our results for the
I50V mutant are also consistent with another report in which
the fitness of this mutant was reduced 90% relative to that of
the wild type as measured in an SCA in combination with the
L10F and M46I mutants (44). Our results are also consistent
with our previously published results for NNRTI drug-resistant
mutants. The fitness of the K103N mutant is similar to that of
the WT, whereas the fitness levels of the V106A, G190S, and
P226L mutants are reduced (4, 24, 28, 48).
In contrast to our study, the replication deficit of the V82A
mutant in an NL4-3 background has been reported to be sim-
ilar to the WT in an SCA, only 20% reduced, as measured by
a chlorophenol red-?-D-galactopyranoside (CPRG)/?-galacto-
side assay (35). However, this study used a different method to
measure the infectivity of the mutants and did not compare the
WT and mutant in the same culture. Competitions where WT
and mutant viruses are in the same culture have been shown to
be more sensitive to differences (12). Therefore, differences in
sensitivity may explain why our SCA may have detected a
greater reduction in fitness for the V82A mutant. In support of
our results, the V82A mutant had reduced fitness in the con-
text of a clinical isolate sequence in the single-cycle Monogram
RC assay, again indicating that the sequence backbone may
influence the results (7). The results of the current study and
previous work further support the idea that the method used to
measure fitness may influence the relative fitness of some mu-
tants. We suspect that these methodological differences can
account for at least some of the contradictory fitness values for
drug-resistant mutants that have been reported in some previ-
ously published studies.
Cong et al. have tested the relative fitness, which was differ-
ent from our results, of several RT mutants using an MCA
(13). There were three main differences between the two assays
used: the vector backbone (NL4-3 versus HXB2), the cell type
VOL. 48, 2010 HIV FITNESS IN SINGLE- AND MULTIPLE-CYCLE ASSAYS4041
(PM1 versus MT4 cells), and the method used to detect the
proportion of viruses (flow cytometry versus sequencing). We
have shown previously that the flow cytometry used in our
study and the sequencing used in the Cong study give similar
relative ratios of mutant to WT virus (21), and the studies
described here show that the relative fitness in MT4 and PM1
cells is the same. The only explanation we could find for the
discordant results is differences in sequence backbones. We
and others have previously shown that the background se-
quence in which a drug-resistant mutant develops can influ-
ence its relative fitness, prevalence in patients, and level of
drug resistance (20, 23, 46, 47).
There are three differences between the SCA and MCA that
may or may not explain the differences in fitness seen for the
protease mutants. These include the use of MLV envelope to
pseudotype the SCA virus stocks, a different transfection re-
agent, and differences in the length of time the virus particles
are in the culture supernatant before they find a target cell.
However, we believe these differences are unlikely explana-
tions for the differences seen between the SCA and MCA,
since they cannot explain the fact that some mutants had
higher relative fitness in the SCA than in the MCA while other
mutants had higher relative fitness in the MCA. Therefore, the
difference would have to be dependent on the specific mutant
being tested. In addition, even though differences in the enve-
lope could impact binding and entry, there is no evidence that
protease activity is needed for binding and entry, and the
transfecting reagents are present during transfection only and
are not present during infection.
The fitness difference between the multiple-cycle and single-
cycle assays for the V82A, I54M, I54L, and D30N mutants
ranges from 39% to 48%. Is this magnitude of difference bio-
logically relevant? Several studies have looked at the correla-
tion of fitness with clinical measures such as viral load and CD4
count. A study of chronically infected subjects using a whole-
virus MCA showed that a 20% decrease in fitness correlates
with at least a 0.5 log10decrease in viral load (8). Therefore,
the mutants identified here, which have about a 40% different
in fitness, could impact the viral load by 1 log10. Another study,
with acutely infected subjects, showed that subjects with an RC
value of ?0.42 measured using an SCA had an average CD4
cell count that was ?100 cells/?l higher than that of subjects
with an RC value of ?0.42 (6). Therefore, mutants such as the
D30N and I54M mutants, which cross the 0.42 threshold, could
have an impact on CD4 cell counts. Another study showed that
a cutoff of 65% for RC predicted better response, such that
subjects with an RC of ?65% had a better outcome than
subjects with an RC of ?65% (14). Again, the difference in
fitness for our mutants crosses this threshold. Other studies,
with elite controllers, whose viral load is never ?2,000 cop-
ies/ml without therapy, show that their viruses have 20% lower
fitness than those of progressors (37, 38). Our group has also
shown that NNRTI-resistant mutants, which have a 30 to 70%
decrease in fitness compared to highly prevalent mutants, are
less prevalent in vivo (24, 28). These studies serve as examples
that the magnitude of the difference we see between the SCA
and the MCA for protease mutants could be clinically impor-
More studies are warranted to determine if fitness as mea-
sured by an SCA or an MCA correlates better with clinical
outcome. Until that is determined, studies of fitness, particu-
larly for protease mutants, warrant the use of both types of
assays. Currently, the Phenosense assay from Monogram Bio-
sciences, a drug resistance assay, is the only assay that also
provides clinicians with an RC value measured using an SCA.
Our work indicates that patient samples containing protease
mutants that are tested by this assay may not have an accurate
fitness measurement. Therefore, clinicians should use this
value cautiously. The magnitude of the difference measured
between the SCA and the MCA is large enough to have a
biological impact in vivo. Since this work is the most compre-
hensive comparison of drug-resistant mutants in both assays to
date, more studies in different clinical settings are needed to
determine its clinical importance.
Our results show that the type of fitness assay that is chosen
to analyze the relative fitness of drug-resistant mutants may
influence the result. Direct comparison of relative fitness using
a multiple-cycle and a single-cycle assay may be a way to
determine whether a particular mutant is likely to affect early
or late steps in the life cycle. Studies are under way to deter-
mine if the early steps of the virus life cycle affect fitness using
both the SCA and MCA and if late steps in the life cycle only
affect fitness using the MCA.
We thank Kyriakos Deriziotis, Dongge Li, Kora Fox, and Xiafang
Liu for their technical expertise.
This work was supported in part by NIH R01-AI-065217 and R01-
AI-041387 and the University of Rochester Developmental Center for
AIDS Research (P30-AI-078498).
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