RT-SHIV subpopulation dynamics in infected macaques during anti-HIV therapy.

Abstract

To study the dynamics of wild-type and drug-resistant HIV-1 RT variants, we developed a methodology that follows the fates of individual genomes over time within the viral quasispecies. Single genome sequences were obtained from 3 pigtail macaques infected with a recombinant simian immunodeficiency virus containing the RT coding region from HIV-1 (RT-SHIV) and treated with short-course efavirenz monotherapy 13 weeks post-infection followed by daily combination antiretroviral therapy (ART) beginning at week 17. Bioinformatics tools were constructed to trace individual genomes from the beginning of infection to the end of the treatment.
A well characterized challenge RT-SHIV inoculum was used to infect three monkeys. The RT-SHIV inoculum had 9 variant subpopulations and the dominant subpopulation accounted for 80% of the total genomes. In two of the three monkeys, the inoculated wild-type virus was rapidly replaced by new wild type variants. By week 13, the original dominant subpopulation in the inoculum was replaced by new dominant subpopulations, followed by emergence of variants carrying known NNRTI resistance mutations. However, during ART, virus subpopulations containing resistance mutations did not outgrow the wide-type subpopulations until a minor subpopulation carrying linked drug resistance mutations (K103N/M184I) emerged. We observed that persistent viremia during ART is primarily made up of wild type subpopulations. We also found that subpopulations carrying the V75L mutation, not known to be associated with NNRTI resistance, emerged initially in week 13 in two macaques. Eventually, all subpopulations from these two macaques carried the V75L mutation.
This study quantitatively describes virus evolution and population dynamics patterns in an animal model. The fact that wild type subpopulations remained as dominant subpopulations during ART treatment suggests that the presence or absence of at least some known drug resistant mutations may not greatly affect virus replication capacity in vivo. Additionally, the emergence and prevalence of V75L indicates that this mutation may provide the virus a selective advantage, perhaps escaping the host immure system surveillance. Our new method to quantitatively analyze viral population dynamics enabled us to observe the relative competitiveness and adaption of different viral variants and provided a valuable tool for studying HIV subpopulation emergence, persistence, and decline during ART.

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BioMed Central
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Retrovirology
Open Access
Research
RT-SHIV subpopulation dynamics in infected macaques during
anti-HIV therapy
Wei Shao*1, Mary Kearney2, Frank Maldarelli2, John W Mellors3,
Robert M Stephens1, Jeffrey D Lifson4, Vineet N KewalRamani2,
Zandrea Ambrose3, John M Coffin5 and Sarah E Palmer2,6
Address: 1Advanced Biomedical Computing Center, SAIC Frederick, Inc, National Cancer Institute at Frederick, Frederick, MD, USA, 2HIV Drug
Resistance Program, NCI, Frederick, MD, USA, 3Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,
4AIDS and Cancer Virus Program, SAIC Frederick, Inc, National Cancer Institute at Frederick, Frederick, MD, USA, 5Tufts University, Boston, MA,
USA and 6Department of Virology, Swedish Institute for Infectious Disease Control and Karolinska Institutet, Stockholm, Sweden
Email: Wei Shao* - shaow@mail.nih.gov; Mary Kearney - kearneym@mail.nih.gov; Frank Maldarelli - fmalli@mail.nih.gov;
John W Mellors - Mellors@dom.pitt.edu; Robert M Stephens - stephensr@mail.nih.gov; Jeffrey D Lifson - Jeffrey.Lifson@nih.gov;
Vineet N KewalRamani - vineet@ncifcrf.gov; Zandrea Ambrose - zaa4@pitt.edu; John M Coffin - coffinj@mail.nih.gov;
Sarah E Palmer - sarah.palmer@smi.se
* Corresponding author
Abstract
Background: To study the dynamics of wild-type and drug-resistant HIV-1 RT variants, we developed a methodology that
follows the fates of individual genomes over time within the viral quasispecies. Single genome sequences were obtained from 3
pigtail macaques infected with a recombinant simian immunodeficiency virus containing the RT coding region from HIV-1 (RT-
SHIV) and treated with short-course efavirenz monotherapy 13 weeks post-infection followed by daily combination
antiretroviral therapy (ART) beginning at week 17. Bioinformatics tools were constructed to trace individual genomes from the
beginning of infection to the end of the treatment.
Results: A well characterized challenge RT-SHIV inoculum was used to infect three monkeys. The RT-SHIV inoculum had 9
variant subpopulations and the dominant subpopulation accounted for 80% of the total genomes. In two of the three monkeys,
the inoculated wild-type virus was rapidly replaced by new wild type variants. By week 13, the original dominant subpopulation
in the inoculum was replaced by new dominant subpopulations, followed by emergence of variants carrying known NNRTI
resistance mutations. However, during ART, virus subpopulations containing resistance mutations did not outgrow the wide-
type subpopulations until a minor subpopulation carrying linked drug resistance mutations (K103N/M184I) emerged. We
observed that persistent viremia during ART is primarily made up of wild type subpopulations. We also found that
subpopulations carrying the V75L mutation, not known to be associated with NNRTI resistance, emerged initially in week 13 in
two macaques. Eventually, all subpopulations from these two macaques carried the V75L mutation.
Conclusion: This study quantitatively describes virus evolution and population dynamics patterns in an animal model. The fact
that wild type subpopulations remained as dominant subpopulations during ART treatment suggests that the presence or
absence of at least some known drug resistant mutations may not greatly affect virus replication capacity in vivo. Additionally,
the emergence and prevalence of V75L indicates that this mutation may provide the virus a selective advantage, perhaps escaping
the host immure system surveillance. Our new method to quantitatively analyze viral population dynamics enabled us to observe
the relative competitiveness and adaption of different viral variants and provided a valuable tool for studying HIV subpopulation
emergence, persistence, and decline during ART.
Published: 4 November 2009
Retrovirology 2009, 6:101 doi:10.1186/1742-4690-6-101
Received: 14 September 2009
Accepted: 4 November 2009
This article is available from: http://www.retrovirology.com/content/6/1/101
© 2009 Shao et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Background
Antiretroviral therapy (ART) suppresses HIV-1 replication
in vivo but does not eradicate the virus. Consequentially,
drug resistance remains a major obstacle to effective ther-
apy [1]. Recent evidence indicates that mucosal transmis-
sion of HIV-1 infection usually involves the establishment
of systemic infection by only a single viral variant [2-5].
After transmission, the virus is able to diversify into com-
plex subpopulations due to its rapid replication cycle and
high mutation rate. In a ten year period, HIV-1 genomes
in an infected patient can be 3000 generations removed
from the initial infecting virus [1]. Understanding HIV
population dynamics and evolution is therefore impor-
tant for understanding AIDS pathogenesis and the emer-
gence of drug resistance mutations [6,7].
The intra-patient evolution of HIV-1 subpopulations can
be shaped by several selective forces, including host
immune surveillance, ART, and competition between dif-
ferent virus variants for host resources [8,9]. A major fac-
tor affecting HIV-1 evolution in treated patients is the
emergence of drug resistant mutations, which have been
reported for all effective antiviral drugs developed to date
[10]. Mutations conferring escape from both humoral and
cellular immune responses are also frequent [11,12]. To
date, there have been few longitudinal studies on the
dynamics of virus subpopulations within infected indi-
viduals, including their emergence, persistence, preva-
lence, and decline during infection and treatment.
Charpentier et al. followed the emergence of drug resist-
ance mutations in patients treated with protease inhibi-
tors and described the dynamics of the major HIV-1
subpopulations [13]. Ball et al proposed a mathematical
model to describe intra-host HIV evolution in terms of
mutation, competition, and strain replacement [14,15].
However, quantitative documentation of virus popula-
tion structure and dynamics during the course of infection
is rare in the literature. One particular difficulty with HIV-
1 in infected patients is that the virus population structure
at the time of infection, and shortly thereafter, cannot be
directly assessed. For this reason, we have analyzed
plasma from macaques infected with a well-defined SIV
chimeric virus containing the RT coding region of HIV-1
(RT-SHIVmne) [16]. In an earlier study, we reported the fre-
quency of drug resistance mutations in virus isolated from
longitudinal plasma samples after infection and during
treatment [17]. We report here the analysis of multiple
single genome sequences to quantify the number of sub-
populations (populations consisting of identical virus
variants) and to analyze the complex dynamics of these
populations during the course of infection and treatment.
Results
Population structures in early stages of RT-SHIV infection
and treatment of animal M03250
HIV-1 RT subpopulation dynamics were analyzed in the
plasma of 3 pigtail macaques infected with RT-SHIV
(Table 1). Samples were obtained from a previous study
aimed at evaluating the effects of prior exposure to NNRTI
monotherapy on subsequent combination ART [17], sim-
ilar to the use of single-dose nevirapine to prevent mother
to child transmission [18-21]. The animals were treated
with a short course of efavirenz (EFV) at week 13, fol-
lowed by daily combination therapy of tenofovir (TNF),
emtracitabine (FTC), and EFV from weeks 17-37 post-
inoculation. Frequent and convenient sampling, access to
the virus inoculum, and lack of adherence issues make the
RT-SHIV macaque model ideal for investigating viral pop-
ulation dynamics prior to initiating therapy, after initiat-
ing short-course monotherapy, and during ART.
Macaque M03250 failed the combination therapy with
the appearance of multidrug resistant virus starting at
week 22, 5 weeks after combination ART was initiated.
Viremia in the other two macaques remained suppressed
during the course of therapy. In each virus population,
dominant and minor subpopulations were found among
the sequences obtained by single-genome sequencing
(SGS) at the time points shown in Table 1. The sequence
of each subpopulation of M03250 was used to construct a
neighbor-joining tree (Figure 1), with subpopulations
from the same week labeled with a symbol of the same
color and shape and each subpopulation represented by a
leaf in the tree. In this animal, RT-SHIV evolved into a very
complex population in which subpopulations from early
time points persisted over the course of infection, while
other subpopulations were lost. Subpopulations contain-
ing the drug resistance mutations K103N (AAC and AAT)
Table 1: Treatment and sampling intervals for the 3 macaques.
Number of samplesa
EFV
←
ART
→
Week of sampling01 1213b
1717.519 2223 2425 26373940
M03250
M04007
M04008
39
39
39
24
12
33
37
20
23
40
38
41
44 41 353337 43 41
32
19 31
a Number of sequences obtained at each time point.
b Week 13 was sampled before EFV treatment.

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formed 5 clusters in the phylogeny (Figure 1 Clusters A-E),
indicating that they emerged independently. The earliest
subpopulations containing the EFV resistance mutation
K103N were observed at week 17 in both clusters A (AAA
to AAC)) and B (AAA to AAT)) and at week 17.5 in clusters
C, D, and E (Figure 1).
Phylogenetic analysis of RT-SHIV subpopulations of macaque M03250
Figure 1
Phylogenetic analysis of RT-SHIV subpopulations of macaque M03250. The left panel is a neighbor-joining tree of all
subpopulations of M03250. Each subpopulation is shown as a single sequence for this tree construction. Subpopulations from
each week are represented by symbols coded with the same color and shape. The internal nodes from which subpopulation
clusters containing drug resistant mutations appeared are marked as clusters A, B, C, D, and E, shown enlarged to the right.
Cluster B
Cluster C
Cluster E
Week0
Week1
Week13
Week17
Week17.5
Week19
Week22
Week23
Week24
Week25
week26
Cluster CCluster C
Cluster DCluster D
Cluster E Cluster E
Cluster A Cluster A
Cluster B Cluster B
0.001 0.001
Clusters A
Cluster D

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Neighbor joining trees were also constructed from all
sequences obtained for each time point. Figure 2 shows
the RT-SHIV population from week 0, week 13 (just prior
to EFV monotherapy), and week 17 from monkey
M03250. Several distinct subpopulations were evident,
some consisting of only one sequence with others com-
prising multiple identical sequences (up to 10). At week 0,
there was one dominant subpopulation (subpopulation
1, solid dark green circles). At week 13, the virus popula-
tion was characterized by two dominant subpopulations,
(subpopulations 2 and 3) each comprising 24% of the
total population (solid green diamonds and solid blue
diamonds) while the remaining 52% comprised minor
subpopulations of unique sequences (Figure 2, hollow
diamonds). However, at week 17 following EFV mono-
therapy, there was only one dominant subpopulation
(subpopulation 3, solid light green squares), although
two members of subpopulation 2 were still present. At the
same time, 6 variants containing K103N (AAC) or K103N
(AAT) were detected, which formed 4 subpopulations.
Subpopulation 5 (solid black squares) comprised 3 virus
sequences while the other 3 each had only one sequence
(hollow colored squares, Figure 2). In all samples ana-
lyzed from each of the infected monkeys, we consistently
found one or two dominant subpopulations, along with
many minor subpopulations.
Subpopulation dynamics in monkey M03250
Figure 3 shows the fates of selected RT-SHIV subpopula-
tions in M03250, expressed as percentages of the whole
viral population at each sampling week and Figure 4
shows the same subpopulations as viral RNA copies/ml
plasma. As shown in Figures 3 and 4, the dominant sub-
population found in the original virus challenge stock
(sub1, week 0) was also the dominant subpopulation in
the first plasma sample collected from M03250 (Figure 3,
sub 1 at week 0 and week 1). This variant, however, was
not found by week 13 as new subpopulations emerged. It
was replaced by two new wild type dominant subpopula-
tions emerging at week 13 prior to EFV treatment (sub2,
24% and sub3, 24%; Figure 3). The frequency of sub2
declined significantly between weeks 13 and 17, and the
two remained relatively constant throughout a 5 weeks
period on combination therapy and a 3-log decline in
viremia, even though neither subpopulation carried any
known drug resistant mutation. They subsequently
became minor species at weeks 23 and 24 (Figure 3).
During the course of infection and treatment, many sub-
populations carrying drug resistant mutations emerged.
However, none became dominant before the emergence
and expansion (to about 75% of the virus population) of
the double mutant K103N/M184I (resistant to both EFV
and FTC), beginning at week 23 and coincident with the
onset of virologic failure. EFV resistance mutations
(K103N) initially were observed at week 17, the first sam-
ple after EFV monotherapy: an AAC allele (sub5, Figures 3
and 4) and an AAT allele (sub6, Figures 3 and 4). The AAC
subpopulation remained minor until week 22, after
which time it became undetectable. The AAT subpopula-
tion was detected at week 17 and never became dominant.
The same was true at weeks 22 and 23 for a variant carry-
ing two drug resistance mutations: K65R/K103N(AAC)
(Figure 3, sub7), encoding resistance to TNF as well as
EFV. Overall, at week 23, 6 weeks after the initiation of
ART, 11 out of 23 subpopulations contained K103N and
4 out of 23 subpopulations contained K65R (3 as K65R/
K103N). Subpopulations with a single K103N mutation
(without linkage to another drug resistance mutation)
were 30% of all viral populations of week 23 (Additional
file 1) and none was the dominant subpopulation (Figure
3). In the neighbor joining tree, subpopulations contain-
ing K103N(AAC)/K65R or K103N(AAC)/M184I and sev-
eral others containing K103N(AAC) formed a cluster.
Several subpopulations containing K103N(AAT) and
K103N(AAC) formed another cluster (Figure 1). In total,
5 of 9 subpopulations contained K103N at week 24, all
existing as minor subpopulations. The subpopulations
containing only K103N were 2.7% of the population at
this week, declining from 30.1% at week 23 (Additional
file 1), a result of the takeover by the doubly resistant sub-
population 8.
The subpopulation that led to virologic failure in this
macaque carried the linked drug resistant mutations
K103N(AAC)/M184I. This species was observed at week
23 as two subpopulations: week 23-26 (sub8) and week
23-32 (Additional file 1), one becoming undetectable the
very next week (week 23-32 in Additional file 1), and one
persisting and leading to virologic failure at weeks 25 and
26 (sub8 in Figures 3 and 4). Interestingly, a wild-type
subpopulation first appeared as a minor species at week
13 (Figure 3, sub4) and became dominant (50%) after
failure of combination therapy in week 24. This subpopu-
lation did not carry any drug resistance mutations, yet it
persisted and even increased about 9-fold in frequency
and 300-fold in terms of its absolute amount over the
course of infection and treatment through week 26 (Fig-
ures 3 and 4).
Another mutation not associated with drug resistance at
position 75 in RT (V75L) was detected first in every sub-
population at week 13 in animal M03250 prior to EFV
treatment. This mutation was also present in almost all
subpopulations at later time points (Additional file 1). It
was not detected by SGS in the challenge stock used to
infect this macaque and was not seen at week 1. It was
possible that this variant existed in the challenge stock at
a frequency below our detection sensitivity by SGS. There-
fore, we used 454 pyrosequencing to increase the sensitiv-

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Structure of RT-SHIV populations from macaque M03250
Figure 2
Structure of RT-SHIV populations from macaque M03250. The tree shows sequences from weeks 0 (circles), 13 (dia-
monds), and 17 (squares). Subpopulations consisting of multiple sequences are marked with solid shapes while subpopulations
consisting of single sequence are marked with hollow shapes. Different subpopulations containing K103N are also labeled with
solid shapes. Subpopulation designations (subpopulations 1, 2, 3, 5, and 6) correspond to those in Figures 3 and 4. Subpopula-
tions not shown in Figures 3 and 4 were not given a subpopulation designation.