Persistence of lamivudine-sensitive HIV-1 quasispecies in the presence of lamivudine in vitro and in vivo.

Abstract

The establishment of persistent infection is one of the major obstacles facing the eradication of HIV-1. To improve our understanding of the mechanisms of viral persistence, we investigated the fate of defined viral quasispecies under conditions that might favor their eradication. We retrospectively analyzed changes in viral populations in HIV-1-infected patients treated with zidovudine/lamivudine and subsequently failing therapy within months in the years 1996 to 1997. Furthermore, we developed an in vitro model based on simultaneous infection of T cells with 2 or more different viral variants. Changes in minority quasispecies of drug-sensitive and drug-resistant HIV-1 variants based on lamivudine and the corresponding lamivudine-resistant viruses carrying the M184I or M184V mutation were investigated using an allele-specific real-time polymerase chain reaction assay. We demonstrate that lamivudine-sensitive and lamivudine-resistant HIV-1 variants are able to persist despite highly unfavorable conditions in vivo and in vitro and that selective advantages of viral variants can vary depending on the complexity of other simultaneously replicating viral variants.

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BASIC SCIENCE
PersistenceofLamivudine-SensitiveHIV-1Quasispeciesin
the Presence of Lamivudine In Vitro and In Vivo
Kristina Allers, PhD Student,* Stefanie A. Knoepfel, PhD Student,* Pia Rauch,* Hauke Walter, MD,*
Milos Opravil, MD,† Marek Fischer, PhD,† Huldrych F. Gu ¨nthard, MD,† and Karin J. Metzner, MD*
Summary: The establishment of persistent infection is one of the
major obstacles facing the eradication of HIV-1. To improve our
understanding of the mechanisms of viral persistence, we investigated
the fate of defined viral quasispecies under conditions that might
favor their eradication. We retrospectively analyzed changes in viral
populations in HIV-1–infected patients treated with zidovudine/
lamivudine and subsequently failing therapy within months in the
years 1996 to 1997. Furthermore, we developed an in vitro model
based on simultaneous infection of T cells with 2 or more different
viral variants. Changes in minority quasispecies of drug-sensitive and
drug-resistant HIV-1 variants based on lamivudine and the
corresponding lamivudine-resistant viruses carrying the M184I or
M184V mutation were investigated using an allele-specific real-time
polymerase chain reaction assay. We demonstrate that lamivudine-
sensitive and lamivudine-resistant HIV-1 variants are able to persist
despite highly unfavorable conditions in vivo and in vitro and that
selective advantages of viral variants can vary depending on the
complexity of other simultaneously replicating viral variants.
Key Words: allele-specific real-time polymerase chain reaction,
HIV-1, M184V, M184I, minority quasispecies, persistence
(J Acquir Immune Defic Syndr 2007;44:377–385)
S
have dramatically decreased.1–3The development of drug
resistance and the inability of current therapeutic strategies to
eradicate the infection are attributable to the characteristics
of HIV-1. First, the dynamics of HIV-1 replication are fast,4,5
and the generated viral population is characterized by high
ince the introduction of antiretroviral therapy (ART), the
morbidity and mortality of patients infected with HIV-1
diversity.6Second, HIV-1 establishes a persistent infection,
and immediately after primary infection, HIV-1 fills up the
viral reservoir.7–10This reservoir has a decay rate of up to 44
months11–13and is constantly replenished by current viral
populations. Thus, newly developed drug-resistant viruses can
enter the reservoir and persist.14It is still not completely
understood how HIV-1 predominantly persists, whether it is
a latent stage or ongoing replication, and how different HIV-1
quasispecies influence each other.10,15–24
To gain more insight into the dynamics of the develop-
ment of drug resistance, replication efficiencies, and persis-
tence of disadvantaged viral quasispecies, we retrospectively
measured changes in viral populations in HIV-1–infected
patients participating in an early, randomized, dual- versus
triple-drug therapeutic study during the years 1996 to 1998
(Early Antiretroviral Therapy in HIV [EARTH]).25Using an
allele-specific real-time polymerase chain reaction (AS-PCR)
assay allowing simultaneous quantification of different HIV-1
variants in the same sample with discriminatory abilities of
0.2% and less, we analyzed changes in lamivudine-sensitive
and lamivudine-resistant HIV-1 variants in those patients
treated with the nucleoside reverse transcriptase (RT) inhibitors
zidovudine and lamivudine and having a continuous detectable
viral load during the follow-up period. Currently, because of
combination therapy with at least 3 different drugs and the
success of prolonged reduction of virus replication to undetect-
able levels, this kind of investigations is not feasible anymore.
Therefore, in vitro assays have been developed to determine
replication efficiencies of certain viral variants. The most
commonly used models for estimating the replicative efficiency
of HIV-1 quasispecies are based on single-round infections,26
assayscomparingthe replication rates inseparatecellcultures,27
and dual-infection/competition assays.28–32To determine the
viral persistence of variants for which the environmental con-
ditions are not favorable, it is necessary to detect and quantify
minority quasispecies of viruses. Therefore, we developed an
in vitro assay specifically designed to track changes in viral
populations after coinfection of T cells with 2 or more different
HIV-1 variants using sensitive AS-PCR. For this approach, we
used lamivudine-sensitive HIV-1 and the well-characterized
lamivudine-resistant viral variants carrying the mutation M184I
or M184V in the viral RT, both of which confer high-level
resistance. Treatment with lamivudine leads almost exclusively
to the development of HIV-1 variants containing the described
changes in codon 184 in vivo and in vitro.33–35We show that in
HIV-1–infected patients treated with zidovudine/lamivudine
and in our cell culture model, the lamivudine-sensitive HIV-1
Received for publication August 21, 2006; accepted December 6, 2006.
From the *Institute of Clinical and Molecular Virology, University of
Erlangen-Nuremberg, Erlangen, Germany; and †Division of Infectious
Diseases and Hospital Epidemiology, Department of Medicine, University
Hospital Zurich, Zurich, Switzerland.
Supported by the Deutsche Forschungsgemeinschaft (SFB 466, Graduierten-
kolleg 1071) and the Wilhelm Sander Stiftung (grant 2002.079.1). H. F.
Gu ¨nthard was supported by the Swiss National Science Foundation (grant
3100AO-103748/1). The Early Antiretroviral Therapy in HIV (EARTH)
Study was financed by grants of the Swiss Federal Office of Public Health
(grants 32-46016.95 and 3600.010.1), the Swiss HIV Cohort Study
(project no 144), GlaxoSmithKline, and Abbott Laboratories.
Reprints: Karin J. Metzner, MD, Institute of Clinical and Molecular Virology,
University of Erlangen-Nuremberg, Schlossgarten 4, D-91054 Erlangen,
Germany (e-mail: karin.metzner@viro.med.uni-erlangen.de).
Copyright ? 2007 by Lippincott Williams & Wilkins
J Acquir Immune Defic Syndr?Volume 44, Number 4, April 1, 2007
377
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Page 2

variantcanpersist byresidualreplicationdespite the presenceof
lamivudine in sufficient inhibitory concentrations and despite
the presence of the lamivudine-resistant quasispecies as the
major viral population. Vice versa, we have observed residual
replication of lamivudine-resistant variants in the presence of
lamivudine-sensitive HIV-1 and in the absence of lamivudine.
In addition, the complexity of simultaneously replicating
viruses has an enormous impact on replication efficiencies of
certain viral quasispecies.
MATERIAL AND METHODS
Study Design and Patients
Four patients from the EARTH study25referred to the
clinical center in Zurich were included in this study, which was
approved by the Ethics Committee of the University Hospital
Zurich. All patients also participated in the Swiss HIV Cohort
Study. Written informed consent was obtained from each
patient before inclusion in this study. Viral RNA from plasma
samples collected from blood in ethylenediaminetetraacetic
acid (EDTA) anticoagulant was isolated, reverse transcribed,
and amplified as previously described.36,37HIV-1 RNA in
plasmawas measured using an adaptation of the Roche (Basel,
Switzerland) Amplicor monitor assay (version 1.5), reaching
a sensitivity of the assay of #50 copies/mL of plasma.38,39
Viruses
The mutations M184I and M184V were separately
introduced into the molecular HIV-1 clone pNL4-3 (AIDS
Research and Reference Reagent Program, Division of AIDS,
National Institute of Allergy and Infectious Diseases [NIAID],
National Institutes of Health [NIH]), and virus stocks were
generated and characterized as previously described.40
Different mixtures of lamivudine-sensitive HIV-1/NL4-3 and
lamivudine-resistant variants HIV-1/NL4-3M184I and HIV-1/
NL4-3M184Vwere used for infections of CEM3174 cells or
peripheral blood mononuclear cells (PBMCs). Calculations of
ratios of lamivudine-sensitive and lamivudine-resistant viral
variants were based on HIV-1 RNA copy numbers. The total
multiplicity of infection (MOI) was approximately 0.001 in all
performed infection and competition experiments.
Cells and Infections
PBMCswere obtained from healthy HIV-1–seronegative
donors (buffy coats from the Institute of Transfusion
Medicine, Suhl, Germany), separated over a Ficoll gradient,
cultivated in RPMI 1640 complete medium (containing 10%
heat-inactivated fetal calf serum, 2 mM of glutamine, 40 mM
of streptomycin, and 170 mM of penicillin), and activated with
3 mg/mL of phytohemagglutinin (PHA; Sigma, Munich,
Germany) and 10 U/mL of recombinant interleukin (IL)-2
(Hoffmann-La Roche, New Jersey, NJ) for 2 days. For dual-
and triple-infection/competition experiments, cells were
infected by spinoculation.41Cells were cultivated in RPMI
1640 complete medium supplemented with 10 U/mL of IL-2.
CEM3174 cells were identically handled, with the exception
that those cells were not treated with PHA and IL-2.
Lamivudine (a gift from GlaxoSmithKline) was added to
the cell cultures in different concentrations (0.09–2.0 mM).
Because of possible variabilities in drug susceptibility in
PBMCs of different donors, lamivudine susceptibility assays
were performed in parallel to the infection and competition
experiments to obtain inhibitory concentrations of 50%, 75%,
and 95% (IC50, IC75, and IC95, respectively) for each donor’s
cells. In experiments with CEM3174 cells, we used 0.09 and
0.8 mM of lamivudine comparable with IC50 and IC95,
respectively (H. Walter, personal communication, 2004).
Supernatants were collected daily or every second day. HIV-1
RNA copy numbers were determined by an in-house viral
load assay, and AS-PCR assays for HIV-1/NL4-3, HIV-1/
NL4-3M184I, and HIV-1/NL4-3M184Vvariants were performed.
Briefly, viral RNA was extracted and reverse transcribed as
previously described.37,42A quantitative real-time PCR assay
was carried out using viral complementary DNA (cDNA),
primers int 4452 5#-GTAGCAGTTCATGTAGCCAGTG-3#
and int 4677 rc 5#-CTCCTTGACTTTGGGGATTGTAG-3#,
and SYBR green (Molecular Probes, Leiden, The Nether-
lands) as described elsewhere.36Evaluation of the discrimi-
natory ability and validation of the M184/V AS-PCR assay
have been described previously.36AS-PCRs of the M184I
mutation and its corresponding lamivudine-sensitive variant
were designed and tested following the same procedures.
Primers used for the M184/I AS-PCRs were pol 2981 5#-
TCAGTACAATGTGCTTCCACAGG-3#, IN 184wt 5#-AGAT
CCTACATACAAATCATIC-3#, and IN M184I 5#-CAGATCC
TACATACAAATCATIT-3#.Amplification,dataacquisition, and
analysis were performed using the ABI Prism 7700 Sequence
Detector System (Applied Biosystems, Foster City, CA). The
estimated discriminatory abilities for the different lamivudine-
sensitive and mutant sequences are as follows: 0.1% for
detection of the M184 lamivudine-sensitive sequence as
a minor population in the presence of M184Vand 0.05% in the
presence of M184I and 0.2% for M184V and M184I,
respectively.
To confirm the presence of guanine at the last position of
codon M184 (ATG), selected amplicons of the wild-type part
of the M184/V AS-PCR assay were sequenced using the
BigDye Terminator version 3.1 kit and the sequencer ABI
PRISM 3100 (Applied Biosystems, Foster City, CA) accord-
ing to the manufacturer’s instructions.
Calculation of Selection Differences of Distinct
HIV-1 Variants and Statistical Analysis
Replication efficiencies of distinct HIV-1 variants were
calculated by a mathematic formula proposed by Goudsmit
et al.43The formulawas applied to our assay design as follows:
s = 1/t ln [q(t)p(0)/p(t)q(0)], where s is the selection difference
between 2 HIV-1 variants q and p. The s values correspond to
changes in replication efficiency (re) when expressed in per-
centages.43To obtain revalues, all s data have been multiplied
by 100%. Positive revalues reflect a selective advantage of
a first HIV-1 variant compared with a second viral variant, and
vice versa. In our dual-infection/competition experiments and
using the patients’ data, q represents the proportion of the
respective lamivudine-resistant HIV-1 variant and p is the
proportion of lamivudine-sensitive virus. In triple-infection/
competition experiments, q is the proportion of HIV-1/
NL4-3M184Vand p represents HIV-1/NL4-3M184I. In regard to
378
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Allers et al
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Page 3

patients’ data, time (0) represents the first day of ART and
(t) represents later time points during therapy in days. In cell
culture experiments, the determination of (0) and (t) was based
on the dynamics of virus replication. Time (0) reflects thevirus
inoculum at day 0 or the first measured viral ratio at day 1 after
infection, and (t) is the time until peak viremia is reached in
days. The 2-tailed Student t test for independent samples was
used for comparing the means of 2 groups of re values.
A P value of ,0.05 was considered as a significant difference
between 2 groups. For linear relations of 2 parameters, the
Pearson correlation coefficient analysis was applied.
RESULTS
Changes in the Composition of HIV-1 Variants
in Patients Receiving Zidovudine/Lamivudine
We have retrospectively investigated changes within the
viral population in patients receiving zidovudine/lamivudine
dual therapy in the years 1996 and 1997 by analyzing the
appearance of lamivudine-resistant variants and the persis-
tence of lamivudine-sensitive virus. All 4 patients showed a
transient reduction in viral load of approximately 1 to 2 logs
withinthefirst8weeksafter theonsetofzidovudine/lamivudine
dual therapy (Fig. 1). Afterward, viral loads increased up to
levels somewhat lower than viral loads at baseline. By
evaluating the composition of viral variants carrying the
M184V mutation or the lamivudine-sensitive M184 sequence,
we observed that the M184V mutation was not detectable at
baseline at levels .0.2% to 0.6% in those patients. Five to 13
weeks after initiation of zidovudine/lamivudine dual therapy,
viruses carrying the M184V mutation represented most of the
viral population. In 2 patients, we have found clear evidence
that the switch from lamivudine-sensitive to lamivudine-
resistant viruses had already occurred in the phase of viral
decay. In patient 119, less than 0.6% of viruses harbored the
M184V mutation before ART, whereas 33.2% 6 4.5% of
viruses carried the M184V mutation 4.7 weeks after initiation
of ART. In the same period, the viral load decreased from
38,389 to 590 HIV-1 RNA copies/mL of plasma. Calculating
the selective advantage of lamivudine-resistant viruses, the
gain in replication efficiency was 16.7% in the presence of
lamivudine. The same pattern was observed in patient 123; the
M184V mutation was already present in 86.7% 6 2.9% of
viruses 5 weeks after the onset of ART, which means an
advantageous replication efficiency of 23.1%. The viral load
decreased from 268,765 to 17,102 HIV-1 RNA copies/mL of
plasma in those 5 weeks. This shows that despite viral decay,
ongoing virus replication led to the fast outgrowth of
FIGURE 1. Kinetics of viral load and
differences in replication efficiencies
of lamivudine-sensitive and lamivu-
dine-resistantviral
patients on zidovudine/lamivudine
dual therapy. HIV-1 RNA in plasma
was measured using an adaptation
of the Roche Amplicor monitor
version 1.5 reaching a sensitivity of
#50 HIV-1 RNA copies/mL of plasma
(black circles). Lamivudine-resistant
variants carrying the M184V muta-
tion and lamivudine-sensitive virus
were detected by AS-PCR. The per-
centage of the viral
carrying the specific mutation was
used to calculate the absolute HIV-1
RNA copies/mL of plasma based on
the corresponding viral load mea-
surement. Copy numbers represent-
ing the lamivudine-sensitive variants
(open triangles) and M184V variants
(open diamonds) are shown. Gray
symbols represent results when only
1 of the 2 viral variants was detect-
able. Numbers represent percent-
agesofthelamivudine-resistant
viral population. Percentage values
below the limit of detectable minor-
ity quasispecies attributable to low
viral load or assay limitations are
printed in italics. Selection differ-
ences (re) for the M184V variant
are shown; the thick gray line
represents the corresponding time used for the calculation of re M184V. Arrows depict those lamivudine-sensitive variants that
have been sequenced. All sequences revealed the presence of the wild-type codon ATG (methionine).
sequencesin
population
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Page 4

lamivudine-resistant viruses in those patients. After the switch
from lamivudine-sensitive to predominantly lamivudine-
resistant viruses, the lamivudine-resistant strain represented
most of theviral population throughout the follow-up period in
all 4 patients. In at least 3 patients, however, the replication of
lamivudine-sensitive HIV-1 was not completely abolished.
Drug-sensitive viruses represented between 0.6% and almost
30% of the viral population in the plasma during the time of
follow-up. The quantitative and qualitative amounts of drug-
sensitive viruses increased in 1 patient between weeks 24 and
32, which is reflected by a selective disadvantage of drug-
resistant variants (re= 24.6%, patient 119; see Fig. 1). This
might be caused by temporary nonadherence to ART. The
selective advantage of viruses carrying the M184V mutation
were, on average, 5.9% 6 0.1% compared with lamivudine-
sensitive virus during the complete follow-up period of 144 to
170 days after the onset of dual therapy assuming that q(0) is
0.2%, which represents the detection limit for the M184V
mutation.
To prove the presence of the drug-sensitive codon ATG
(methionine) at position 184 and the absence of the drug-
resistant codon ATA (isoleucine), the wild-type specific
amplicon of the AS-PCR of certain patients’ samples was
sequenced for wild-type M184. Sequences revealed the
presence of the drug-sensitive codon ATG (see Fig. 1).
Differences in Replication Efficiencies of
Distinct HIV-1 Variants and Persistence of
Disadvantaged HIV-1 Variants in
Dual-Infection/Competition Experiments
To establish an in vitro model for viral persistence, we
began with the dual infection of CEM3174 with different
ratios of lamivudine-sensitive virus and the respective
lamivudine-resistant variants HIV-1/NL4-3M184Vand HIV-1/
NL4-3M184I. Cells were cultivated in the absence or presence
of lamivudine, and passages to uninfected cells were under-
taken when cytopathic effects were observed (between days
3 and 6 after infection). Frequently harvested supernatants
were analyzed by AS-PCR, and selective differences (re) were
calculated for lamivudine-resistant HIV-1 variants in compar-
ison with lamivudine-sensitive virus. In the absence of
lamivudine, the drug-resistant variant HIV-1/NL4-3M184Ihas
a slight but insignificant disadvantage in its replication
efficiency (Table 1). In the presence of 0.09 mM of lamivudine
(;IC50), we obtained significantly higher revalues for both
lamivudine-resistant variants, which was even more apparent
using 0.8 mM of lamivudine (;IC95). We have not obtained
significant differences in replication efficiencies comparing
the respective revalues of both lamivudine-resistant variants
(P values not shown). In addition, no linear correlation
between revalues and initial ratios of lamivudine-sensitive and
lamivudine-resistant viral variants has been found, suggesting
that the initial amount of the more replication-competent virus
is not decisive for the differences in replication efficiencies. In
all dual-infection/competition experiments in the presence of
0.09 mM of lamivudine, we observed the persistence of
lamivudine-sensitive virus at a range of 1.1% to 46.8%
depending on the initial ratio of lamivudine-sensitive to
lamivudine-resistant viruses (see Table 1). Moreover, even in
the presence of 0.8 mM of lamivudine, drug-sensitive HIV-1
was able to persist when this quasispecies represented most of
the viruses at day 0.
We expanded our dual-infection/competition assay using
PBMCs to reflect the situation in natural target cells of HIV-1.
In contrast to the experiments described previously, passages
of viruses have not been performed. Lamivudine was used in
concentrations of 0.2, 0.6, and 2 mM, respectively, to cover
a broad range of inhibitory concentrations. In parallel to the
infection and competition experiments, lamivudine suscepti-
bility assays were performed to obtain the IC50, IC75, and IC95
for each donor’s cells. The values for IC50were in the range of
0.133 to 0.218 mM of lamivudine (mean: 0.168 6 0.040 mM),
and those for IC75were reached using lamivudine at a con-
centration of 0.235 to 0.358 mM (mean: 0.307 6 0.050 mM).
We were not able to determine the IC95because of the limit of
the p24 ELISA used; the background was too high and the
maximum virus replication in the absence of lamivudine was
approximately 85% to 90% greater than background. The con-
centrations obtained for IC50and IC75suggest that 2 mM of
lamivudine probably widely exceeds the IC95in PBMCs of all
used donors, however.
In PBMCs, the disadvantaged replication efficiencies of
lamivudine-resistantHIV-1variantscomparedwithlamivudine-
sensitive virus in the absence of lamivudine were 28.6% 6
5.1% for HIV-1/NL4-3M184Vand 211.4% 6 4.4% for HIV-1/
NL4-3M184I(see Table 1). Those revalues are lower compared
with the corresponding revalues obtained by dual-infection/
competition experiments using CEM3174 cells, which were
significant in the case of HIV-1/NL4-3M184V(P = 0.024). In
accordance with results from the dual-infection/competition
experiments using CEM3174 cells, rising concentrations of
lamivudine correlate with increasing selection benefits of the
lamivudine-resistant viruses. Complete inhibition of replica-
tion of lamivudine-sensitive virus could not be observed in
PBMCs of different donors, however, not even in the presence
of 2 mM of lamivudine, which corresponds to an IC95.
Analogous to our results using CEM3174 cells, we have not
obtained any significant differences by comparing the
respective revalues of both lamivudine-resistant variants in
PBMCs (P values not shown). Also, no linear correlation has
been found between revalues and initial ratios of lamivudine-
sensitive and lamivudine-resistant viral variants or donor
dependencies, respectively.
Differences in Replication Efficiencies of
Distinct HIV-1 Variants and Ongoing
Replication of Disadvantaged HIV-1 Variants
In Vitro in Triple-Infection/Competition
Experiments
It is assumed that both lamivudine-resistant variants are
already present as minority quasispecies in drug-naive patients
and that viruses carrying the M184I mutation are present in
a higher proportion thanviruses carrying the M184V mutation
because of the favored G-to-A transition (M184V: A-to-G
transition, M184I: G-to-A transition).44Thus, it is understood
that the M184I mutation appears first after the onset of
380
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Allers et al
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Page 5

antiretroviral regimens containing lamivudine and is later
outgrown by the more replication-competent viral variant
carrying the M184V mutation. We transferred our cell culture
model to those possible in vivo situations and infected
CEM3174 cells and PBMCs, respectively, with a mixture
of 3 different viruses—HIV-1/NL4-3, HIV-1/NL4-3M184V, and
HIV-1/NL4-3M184I—in triple-infection/competition assays.
CEM3174 cells were cultured in the presence of
0.8 mM of lamivudine (;IC95) after infection with all 3 viruses.
In all experiments, the lamivudine-sensitive variants repre-
sented most of the viruses used for the infection. In 3 of 4
experiments, we used HIV-1/NL4-3M184I in higher initial
frequencies than HIV-1/NL4-3M184V(Figs. 2A, B, D). Differ-
ences in replication efficiencies of HIV-1/NL4-3M184V have
been calculated compared with HIV-1/NL4-3M184I. In 3 of 4
experiments, the HIV-1/NL4-3M184I variant initially showed
a higher replication efficiency compared with HIV-1/
NL4-3M184V(see Figs. 2A–C). Calculations of re M184Vvalues,
TABLE 1. Differences in res of Lamivudine-Resistant Viruses HIV-1 NL4-3M184V(A) and HIV-1 NL4-3M184I(B), Respectively,
Compared With Lamivudine-Sensitive HIV-1 NL4-3 in Dual-Infection/Competition Experiments Using CEM3174 Cells and Their
Dependencies on Lamivudine Concentrations
A. CEM3174 CellsB. CEM3174 Cells
Lamivudine Concentration (mM)M184(0)–M184(t) (%)re M184V(%)P M184(0)–M184(t) (%)re M184I(%)P
0
0
0
86.1–85.5
41.5–28.1
7.8–6.5
0.3
3.0
0.9
1.4 6 1.4
90.3–95.2
40.7–50.5
12.6–51.5
23.8
21.9
210.0
25.2 6 4.2 Mean 6 SD
,0.001*
0.001†
0.01*
,0.001†
0.09 (;IC50)
0.09 (;IC50)
0.09 (;IC50)
Mean 6 SD
0.8 (;IC95)
0.8 (;IC95)
0.8 (;IC95)
Mean 6 SD
86.1–46.1
41.5–7.2
7.8–1.1
9.4
11.1
10.2
10.2 6 0.9
42.2
50.5
34.1
42.3 6 8.2
90.3–46.8
40.7–15.6
12.6–3.3
11.8
6.6
7.1
8.5 6 2.9
42.4
48.2
37.7
42.8 6 5.3
0.003†
,0.001†
86.1–0.1
41.5–0.1‡
7.8–0.1‡
90.3–0.1
40.7–0.05
12.6–0.05‡
A. PBMCsB. PBMCs
Lamivudine Concentration (mM)Donor M184(0)–M184(t) (%)re M184V(%)P M184(0)–M184(t) (%)re M184I(%)P
0
0
0
0
A 56.1–73.1
2.8–5.5
56.1–64.6
2.8–3.1
210.7
214.2
27.1
22.2
28.6 6 5.1
96.5–98.9
42.4–64.7
96.5–97.8
42.4–58.5
216.7
213.0
26.7
29.3
211.4 6 4.4
B
Mean 6 SD
,0.001*
,0.001†
,0.001§
,0.001*
,0.001†
,0.001§
0.2 (;IC50)
0.2 (;IC50)
0.2 (;IC50)
0.2 (;IC50)
Mean 6 SD
A 56.1–4.4
2.8–0.5
56.1–6.9
2.8–0.6
47.4
25.6
40.8
32.8
36.7 6 9.5
96.5–6.1
42.4–3.1
96.5–64.9
42.4–2.1
67.3
44.6
30.2
39.5
45.4 6 15.8
B
0.065†
0.010§
0.035†
0.012§
0.6 (.IC75)
0.6 (.IC75)
0.6 (.IC75)
0.6 (.IC75)
Mean 6 SD
2 (.IC95)
2 (.IC95)
2 (.IC95)
2 (.IC95)
Mean 6 SD
A56.1–0.7
2.8–0.2
56.1–1.0
2.8–0.2
58.1
37.2
69.1
55.5
55.0 6 13.2
92.9
55.9
90.1
62.7
75.4 6 18.8
96.5–1.7
42.4–0.3
96.5–8.1
42.4–0.6
67.0
59.7
82.3
96.9
76.5 6 16.6
74.1
73.3
108.4
89.9
86.4 6 16.5
B
0.126§0.428§
A 56.1–0.2
2.8–0.2
56.1–0.2
2.8–0.1
96.5–0.8
42.4–0.1
96.5–1.4
42.4–0.1
B
*P value as compared with 0.09 mM of lamivudine (CEM3174 cells) or 0.2 mM of lamivudine (PBMCs), calculated using the Student t test.
†P value as compared with 0.8 mM of lamivudine (CEM3174 cells) or 0.6 mM of lamivudine (PBMCs), calculated using the Student? t test.
‡Values are greater than or less than the detection limits of the M184V and M184I AS-PCR assays; re M184Vis estimated based on those values.
§P value as compared with 2 mM of lamivudine (PBMCs), calculated using the Student t test.
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Page 6

however, including the complete time period of the experi-
ments, revealed that the HIV-1/NL4-3M184Vvariant has a clear
advantage in its replication efficiency compared with HIV-1/
NL4-3M184I. Although, the total re M184V value was slightly
negativein 1 of 4 experiments (re M184V= 22.3%; see Fig. 2B),
the re M184V value of the second part of this experiment
reflected an advantage in the replication efficiency of the
HIV-1/NL4-3M184Vvariant (re M184V= 4.2%). Notably, all dis-
advantaged viral variants persisted as minor populations in all
triple-infection/competition experiments using CEM3174 cells
in the presence of 0.8 mM of lamivudine, except in one
experiment in which lamivudine-sensitive virus was not detect-
able after day 14 (see Fig. 2C).
In corresponding triple-infection/competition experi-
ments, PBMCs were infected with defined ratios of HIV-1/
NL4-3M184V
and HIV-1/NL4-3M184I and cultivated in the
presence of 2 mM (.IC95) of lamivudine for approximately
7 to 9 days. The lamivudine-sensitive variant also represented
the majority in all experiments. The initial ratios of HIV-1/
NL4-3M184V and HIV-1/NL4-3M184I used for infection of
PBMCs ranged from 1:5.8 to 39.2:1 (Table 2). We observed
re M184Vvalues varying from 266.6% to 69.0 % (see Table 2).
In only 2 of 15 experiments was the selective advantage in
favor of the HIV-1/NL4-3M184Ivariant. This was the case in 2
of 4 experiments in which the initial amount of this variant was
higher than the fraction of the HIV-1/NL4-3M184Vvariant. The
HIV-1/NL4-3M184V variant showed a selective advantage,
however, despite a lower initial frequency in the other 2 experi-
ments. Consistent with the dual-infection/competition experi-
ments, we have not found a linear correlation of revalues with
initial ratios of distinct viral variants or variations attributable
to PBMCs of different donors. In almost all triple-infection/
competition experiments using PBMCs, complete inhibition
of replication was not observed for lamivudine-sensitive virus
or the disadvantaged lamivudine-resistant variant.
DISCUSSION
The establishment of persistent infection and the emer-
gence of drug-resistant variants are major obstacles facing the
eradication of HIV-1. Numerous studies have shown that ar-
chived drug-sensitive viruses rapidly reappear after treatment
interruption as a result of therapy failure,45because drug resis-
tance is often associated with reduced replication efficiency.46
It is still not completely understood whether disadvantaged
viral variants persist by latency (ie, long-lived cells harboring
nonreplicating but potentially infectious virus) or by residual
replication. One study that specifically addressed this issue
demonstrated that both mechanisms probably contribute to the
persistence of HIV-1.21Persistence and distinct replication
FIGURE 2. Changes in ratios of
lamivudine-sensitive–to–lamivudine-
resistant viruses in triple-infection/
competitionexperiments
CEM3174 cells. Cells were infected
with different ratios of 3 viruses—
HIV-1/NL4-3, HIV-1/NL4-3M184V, and
HIV-1/NL4-3M184I–and cultivated in
the presence of 0.8 mM of lamivu-
dine, which equals an IC95. Percen-
tagesof HIV-1/NL4-3
triangles), HIV-1/NL4-3M184V (open
diamonds),and
(open squares) were estimated by
AS-PCR.Standard
within the data points; thus, they
are not shown. Selection differences
(re) of HIV-1/NL4-3M184V compared
with HIV-1/NL4-3M184Iare depicted;
the thick gray line represents the
corresponding time used for the
calculation of re M184V.
using
(open
HIV-1/NL4-3M184I
deviations are
382
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Page 7

efficiencies of HIV-1 quasispecies are closely related issues
influenced by multiple factors; for instance, composition of
viral quasispecies, selective pressures, and anatomic sites of
virus replication.
We have addressed both issues by retrospective analysis
of changes in the composition of viral variants in patients
receiving zidovudine/lamivudine dual therapy in the years
1996 and 1997, which did not suppress virus replication
efficiently.25The selection of lamivudine-resistant viruses
occurred rapidly. Notably, in 50% of those patients, the
appearance of lamivudine-resistant viruses had already
occurred during viral decay within the first weeks after
initiation of ART. Lamivudine-sensitive virus persisted as
a substantial fraction of the viral population in the plasma in
almost all patients. Plasmaviruses are described as the actively
replicating viral quasispecies at that specific time point,
suggesting that the persistence of the disadvantaged viral
quasispecies in those patients occurs by residual replication
despite suppressive ART. We cannot exclude the possibility
that the lamivudine-sensitiveviral population was produced by
cells that were infected before the initiation of ART, and
therefore would not have been affected by the RT inhibitor
lamivudine. This seems less likely, however, taking into
account the large amount of lamivudine-sensitive viruses in
those patients and the long follow-up period. It is possible that
those lamivudine-sensitive viruses contained mutations con-
ferring zidovudine resistance, thus having a selective advan-
tage in the context of zidovudine/lamivudine dual therapy.
Thiswould notchange the fact that theseviruseswere sensitive
to lamivudine, however. Therefore, we provide clear evidence
that viruses can persist by residual replication despite their
selective disadvantages in patients receiving ART, which does
not completely abolish virus replication.
The performed in vivo analysis is impractical currently
because of undetectable viral loads in patients receiving
standard triple therapy. Therefore, we have established an
in vitro model based on simultaneous infection of T-cell lines
and primary T cells by 2 or 3 different viral quasispecies
combined with highly sensitive tracking of the fate of viral
variants with reduced replication efficiencies using AS-PCR.
This technology allows, among other things, accurate
estimations of replication efficiencies and investigations of
mechanisms of persistence. In the dual-infection/competition
experiments using CEM3174 cells, we have observed only
a minimal reduction in replication efficiencies of both
lamivudine-resistant variants as compared with drug-sensitive
virus in the absence of lamivudine. Clear disadvantages of
both lamivudine-resistant variants were seen in PBMCs. This
is in accordance with previous results and could be explained
by lower deoxynucleoside triphosphate (dNTP) concentrations
in PBMCs as compared with cell lines.47Alternatively, other
cell-specific factors may exist that influence the process of
reverse transcription. The level of selective advantage of both
lamivudine-resistant variants was positively correlated with
the concentration of lamivudine in CEM3174 cells and
PBMCs.
It has been demonstrated that viruses carrying the
M184I mutation are rapidly outcompeted by viruses harboring
the M184V mutation in vivo.48We have not observed
significant selection differences between both viruses carrying
the M184V or M184I mutation, respectively, in the dual-
infection/competition experiments using CEM3174 cells or
PBMCs, however. In contrast, our data obtained by triple-
infection/competition experiments containing all 3 viral quasi-
species revealed that HIV-1/NL4-3M184Vhad a clear selective
advantage compared with HIV-1/NL4-3M184I in CEM3174
cells and PBMCs. In only 2 of 15 triple-infection/competition
experiments using PBMCs did viruses containing the M184V
drop below the limit of detection, and HIV-1/NL4-3M184Iwas
the selected viral variant. One possible explanation maybe that
TABLE 2. Differences in res of Lamivudine-Resistant Virus HIV-1 NL4-3M184VCompared With HIV-1 NL4-3M184Iin
Triple-Infection/Competition Experiments Using Different Ratios of Lamivudine-Resistant and -Sensitive Viruses for the Infection
Of PBMCs Cultivated in the Presence of 2 mM of Lamivudine (.IC95)
Donor M184V(0)–M184V(t) (%)M184I(0)–M184I(t) (%)re M184V(%) Initial Ratio M184V/M184I
C
D
D
C
D
A
B
C
A
B
C
A
B
A
B
2.6–0.2*
2.6–54.8
5.1–0.2*
5.1–99.6
1.3–99.9*
8.8–99.4
8.8–98.9
8.6–99.9*
7.5–99.8
7.5–99.8
18.9–99.9*
45.3–96.8
45.3–93.4
31.6–99.8
31.6–99.8
15.3–99.9
15.3–36.6
6.3–99.9*
6.3–0.3
1.3–0.2*
3.2–0.3
3.2–1.2
2.5–0.4
0.7–0.4
0.7–0.5
1.3–0.2*
3.1–4.5
3.1–5.4
0.8–0.2*
0.8–0.5
249.5
24.0
266.6
67.2
69.0
52.5
48.8
46.8
35.5
33.0
39.6
1:5.8
1:5.8
1:1.2
1:1.2
1:1
2.8:1
2.8:1
3.4:1
10.6:1
10.6:1
14.1:1
14.4:1
14.4:1
39.2:1
39.2:1
5.7
3.7
28.3
24.4
*Values are greater than or less than the detection limits of the M184V and M184I AS-PCR assays; re M184Vis estimated based on those values.
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Page 8

there was no infectious virus harboring the M184V mutation at
the beginning of the culture. This suggests that the influence of
different viral quasispecies on the replication efficiencies of
each separate variant is high. That is also in accordance to our
data, which were obtained by measuring the selection
differences of viruses harboring the M184V mutation in
HIV-1–infected patients receiving zidovudine/lamivudine dual
therapy. In vivo, the level of selective advantage of the
lamivudine-resistant variant M184V is lower as compared
with our results in the dual-infection/competition experiments,
but it is almost equal in comparison to selection coefficients
obtained in the triple-infection/competition experiments. This
means, in the in vivo situation, that it is rather a comparison of
distinct viruses carrying the M184V mutation with a huge
variety of viruses detected as drug-sensitive variants. For
instance, it is likely that drug resistance against the second
drug, zidovudine, is influencing the replication efficiencies of
viruses in those patients. It has recently been shown that the
diversity of quasispecies of polioviruses has an impact on
population dynamics and pathogenesis.49
Persistenceofviralvariantswithreducedreplicationeffi-
ciencies was observed in almost all of our experiments, even
when using lamivudine in high concentrations synonymous
with at least an IC95. HIV-1 RNA of those viruses in cell-free
supernatants of cell cultures was detectable throughout the
follow-up period. This suggests that viruses persist by residual
replication. We cannot completely exclude the possibility that
the persistence of those viruses in CEM3174 cells was
attributable to infections using the T-cell line adapted HIV-1
strain NL4-3. We found that disadvantaged HIV-1 variants can
also persist in PBMCs from different donors, however.
It is possible that persistence of drug-sensitive viruses
during ART is caused by the simultaneous infection of cells
with drug-sensitive and drug-resistant viruses and that persis-
tence results from complementation between those viruses.
Whether cells can be coinfected with wild-type and drug-
resistant strains, and if so, whether the number of coinfected
cells correlates with the degree of persistence of disadvantaged
viral variants are questions that need further investigation.
At any given time, the dominant majority quasispecies
reflects the best-adapted viral population under specific
circumstances. Alterations within this population can occur
quickly in response to changing environmental conditions, and
viral persistence in the form of viral latency and residual virus
replication contributes to the large pool of different HIV-1
quasispecies. We show for the first time that lamivudine-
sensitive and lamivudine-resistant HIV-1 variants are able to
persist by residual replication in vivo and in vitro despite
highly unfavorable conditions and that replication efficiencies
are not only dependent on cell type or drug concentration
but on the complexity of simultaneously replicating viral
variants. This might have an impact on interpretations of
results obtained by in vitro dual-infection/competition fitness
assays. Further studies are necessary to gain more insight into
the dynamics of viral quasispecies in correlation to the
complexity of the genetic diversity of HIV-1. Competition
experiments combined with sensitive methods to track certain
viral quasispecies, such as AS-PCR, should facilitate such
studies.
ACKNOWLEDGMENTS
The authors are grateful to all the patients who
participated in this study and for their contribution toward
a better understanding of HIV-1 pathogenesis. Moreover, they
thank Bernhard Fleckenstein for his constant support. The
pNL4-3 and IL-2 were kindly provided by the AIDS Research
and Reference Reagent Program Division of AIDS, NIAID,
NIH (M. Martin and Hoffmann-La Roche, respectively), and
lamivudine was kindly provided by GlaxoSmithKline.
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