JOURNAL OF VIROLOGY, Oct. 2010, p. 10820–10831
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 20
Gag-Protease-Mediated Replication Capacity in HIV-1 Subtype
C Chronic Infection: Associations with HLA Type
and Clinical Parameters?†
Jaclyn K. Wright,1Zabrina L. Brumme,2,3Jonathan M. Carlson,4David Heckerman,4Carl M. Kadie,4
Chanson J. Brumme,3Bingxia Wang,5Elena Losina,5Toshiyuki Miura,6Fundisiwe Chonco,1
Mary van der Stok,1Zenele Mncube,1Karen Bishop,1Philip J. R. Goulder,1,7,8
Bruce D. Walker,1,8,9Mark A. Brockman,2,3and Thumbi Ndung’u1,8*
HIV Pathogenesis Programme, Doris Duke Medical Research Institute, Nelson R. Mandela School of Medicine, University of
KwaZulu-Natal, Durban, South Africa1; Simon Fraser University, Burnaby, Canada2; BC Centre for Excellence in HIV/AIDS,
Vancouver, Canada3; eScience Group, Microsoft Research, Redmond, Washington4; Program in HIV Outcomes Research,
Massachusetts General Hospital, Boston, Massachusetts5; Institute of Medical Science, University of Tokyo, Tokyo,
Japan6; Department of Paediatrics, Nuffield Department of Medicine, University of Oxford, Oxford,
United Kingdom7; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of
Technology, and Harvard University, Boston, Massachusetts8; and
Howard Hughes Medical Institute, Chevy Chase, Maryland9
Received 19 May 2010/Accepted 29 July 2010
The mechanisms underlying HIV-1 control by protective HLA class I alleles are not fully understood and
could involve selection of escape mutations in functionally important Gag epitopes resulting in fitness costs.
This study was undertaken to investigate, at the population level, the impact of HLA-mediated immune
pressure in Gag on viral fitness and its influence on HIV-1 pathogenesis. Replication capacities of 406
recombinant viruses encoding plasma-derived Gag-protease from patients chronically infected with HIV-1
subtype C were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. Viral replication
capacities varied significantly with respect to the specific HLA-B alleles expressed by the patient, and protective
HLA-B alleles, most notably HLA-B*81, were associated with lower replication capacities. HLA-associated
mutations at low-entropy sites, especially the HLA-B*81-associated 186S mutation in the TL9 epitope, were
associated with lower replication capacities. Most mutations linked to alterations in replication capacity in the
conserved p24 region decreased replication capacity, while most in the highly variable p17 region increased
replication capacity. Replication capacity also correlated positively with baseline viral load and negatively with
baseline CD4 count but did not correlate with the subsequent rate of CD4 decline. In conclusion, there is
evidence that protective HLA alleles, in particular HLA-B*81, significantly influence Gag-protease function by
driving sequence changes in Gag and that conserved regions of Gag should be included in a vaccine aiming to
drive HIV-1 toward a less fit state. However, the long-term clinical benefit of immune-driven fitness costs is
uncertain given the lack of correlation with longitudinal markers of disease progression.
There is broad heterogeneity in the ability of HIV-infected
individuals to control virus replication, ranging from elite con-
trollers, who maintain undetectable viral loads without treat-
ment, to rapid progressors, who progress to AIDS within 2
years of infection (9, 22, 32). Many interrelated factors, includ-
ing host and viral genetic factors involved in antiviral immunity
and the viral life cycle, may partially account for the differences
in the course of disease progression (10, 11, 30, 41). The
complex interplay between host genetic factors and viral fac-
tors is exemplified by human leukocyte antigen (HLA) class
I-restricted cytotoxic T-lymphocyte (CTL) responses, which
exert considerable immune pressure on the virus, resulting in
escape mutations that affect the interaction of viral and host
proteins, thereby influencing infection outcome.
The exact mechanisms by which some HLA class I alleles,
such as HLA-B*57 and HLA-B*27, are associated with slower
progression to AIDS, while others, such as B*5802 and B*18,
are associated with accelerated disease progression (6, 20, 42),
are unclear. The magnitude and/or breadth of HLA-restricted
CTL responses to the conserved Gag protein has been corre-
lated inversely with disease progression or markers of disease
progression in several studies (12, 21, 28, 31, 35, 43, 46), al-
though there are some exceptions (4, 16, 37), while preferential
targeting of the highly variable envelope protein (as occurs in
HLA-B*5802-positive individuals) correlates with higher viral
loads (21, 29). Protective HLA alleles restrict CTL responses
that impose a strong selection pressure on a few specific Gag
p24 epitopes, resulting in escape mutations (14) for which
fitness costs have been demonstrated either through site-di-
rected mutations introduced into a reference strain back-
ground (2, 8, 25, 38) or through in vivo reversion of these
mutations after transmission to an HLA-mismatched individ-
* Corresponding author. Mailing address: HIV Pathogenesis Pro-
gramme, University of KwaZulu-Natal, 719 Umbilo Road, Durban
4013, South Africa. Phone: 27 31 260 4727. Fax: 27 31 260 4623.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 11 August 2010.
ual (8, 24). Recent evidence suggests that Gag escape muta-
tions with a fitness cost, particularly those in p24, are a signif-
icant determinant of disease progression: the transmitted
number of HLA-B-associated polymorphisms in Gag was
found to significantly impact the viral set point in recipients
(although an associated fitness cost was not shown) (7, 15), and
in a small number of infants, decreased fitness of the transmit-
ted virus with HLA-B*5703/5801-selected mutations in Gag
p24 epitopes resulted in slower disease progression (33, 39).
Also, the number of reverting Gag mutations (thought to re-
vert as a consequence of fitness costs) associated with individ-
ual HLA-B alleles was strongly correlated with the HLA-
linked viral set point in chronically infected patients (26). A
recent in vitro study showed that HLA-associated variation in
Gag-protease, with resulting reduced replication capacity, may
contribute to viral control in HIV-1 subtype B-infected elite
controllers (27). Taken together, these studies suggest that
CTL responses restricted by favorable HLA alleles select for
escape mutations in conserved epitopes, particularly those in
Gag, resulting in a fitness cost to HIV and therefore at least
partly explaining the slower disease progression in individuals
carrying these alleles.
To date, many of the studies investigating the fitness cost of
Gag escape mutations and their clinical relevance have con-
centrated on escape mutations associated with protective HLA
alleles, have not assessed fitness consequences in the natural
sequence background (in the presence of other escape and
compensatory mutations), and/or have focused on a limited
number of patients. Most importantly, the majority of studies
have focused on HIV-1 subtype B. The present study is the first
to use a large population-based approach and clinically derived
Gag-protease sequences to investigate comprehensively the
relationships between immune-driven sequence variation in
Gag, viral replication capacity, and markers of disease progres-
sion in chronic infection with HIV-1 subtype C, the most pre-
dominant subtype in the epidemic. We assayed the replication
capacity of recombinant viruses encoding patient Gag-protease
in an HIV-1-inducible green fluorescent protein (GFP) re-
porter cell line and found associations between lower replica-
tion capacities, protective HLA alleles, protective HLA-asso-
ciated mutations, lower baseline viral loads, and higher
baseline CD4 counts. However, Gag-protease replication ca-
pacity did not correlate with the subsequent rate of CD4 de-
MATERIALS AND METHODS
Study subjects. The study subjects included 406 antiretroviral-naïve individu-
als chronically infected with HIV-1 subtype C from the Sinikithemba cohort in
Durban, South Africa. These individuals were HLA typed to 4-digit resolution by
molecular methods (20). Viral load (Roche Amplicor assay, version 1.5) and
CD4 count (Trucount technology) measurements were obtained at study entry
(baseline) for all participants and at 3-month intervals thereafter for 339 of the
participants (20). At baseline, the median viral load of the cohort was 4.77 log10
HIV RNA copies/ml (interquartile range [IQR], 4.15 to 5.27 log10HIV RNA
copies/ml), and the median CD4 count was 340 cells/mm3(IQR, 238 to 477
cells/mm3). Over the subsequent course of study follow-up (the mean follow-up
time was 2.28 years per individual; IQR, 1.21 to 3.02 years), the median rate of
CD4 decline was ?30 cells/mm3per year (IQR, ?73 to ?3 cells/mm3per year).
The median age of the study subjects at baseline was 31 years (IQR, 27 to 36
years), and 322 (79%) patients were female. Among the study participants, there
was no significant association between age, gender, and baseline viral load or
CD4 count as was reported previously (42), and therefore we did not control for
these variables in analyses. Written informed consent was obtained from all study
subjects, and the study protocol was approved by the Biomedical Research Ethics
Committee of the University of KwaZulu-Natal.
Generation of Gag-protease NL4-3 recombinant virus stocks. Patient Gag-
protease was isolated and inserted into an NL4-3 backbone to generate recom-
binant viruses. Protease was included to maintain the important interaction
between Gag and protease, namely, cleavage of the Gag polyprotein by protease.
Viral RNA was isolated from plasma by use of a QIAamp Viral RNA Mini kit
from Qiagen (Valencia). Reverse transcription-PCR (RT-PCR) was performed
as previously described (27), using a Superscript III One-Step RT-PCR kit
(Invitrogen, Carlsbad, CA) and the following Gag-protease-specific primers: 5?
CAC TGC TTA AGC CTC AAT AAA GCT TGC C 3? (HXB2 nucleotides 512
to 539) and 5? TTT AAC CCT GCT GGG TGT GGT ATY CCT 3? (nucleotides
2851 to 2825). A second round of PCR was performed with 100-mer forward and
reverse primers that were exactly complementary to NL4-3 on either side of
Gag-protease, using a TaKaRa Ex Taq HS enzyme kit (Takara, Shiga, Japan).
Two 50-?l PCR mixtures were prepared for each sample, comprising 37 ?l
diethyl pyrocarbonate (DEPC)-treated water, 5 ?l 10? Ex Taq buffer, 4 ?l of
deoxynucleoside triphosphates (dNTPs), 0.8 ?l forward primer (10 ?M), 0.8 ?l
reverse primer (10 ?M), 0.25 ?l Ex Taq, and 2 ?l RT-PCR product. Thermo-
cycler conditions were as follows: 94°C for 2 min; 40 cycles of 94°C for 30 s, 60°C
for 30 s, and 72°C for 2 min; and 72°C for 7 min. PCR products from two 50-?l
reaction mixtures were pooled, and 10 ?l was set aside for sequencing. The
remainder was used in the generation of recombinant viruses. Gag-protease-
deleted pNL4-3 plasmid was prepared as previously described (27), and large
stocks of the plasmid were generated using a Plasmid Maxi kit (Qiagen, Valencia,
CA). The plasmid was digested for 2 h at 60°C immediately prior to cotransfec-
tion of 2 ? 106Tat-inducible GFP reporter GXR T cells (3) in R10 medium (800
?l RPMI-1640 [Sigma, St. Louis, MO] supplemented with 10% fetal bovine
serum [Gibco, NY], 2 mM L-glutamine [Sigma], 10 mM HEPES [Gibco], and 50
U/ml penicillin-streptomycin [Gibco]) with 10 ?g digested plasmid and ?85 ?l
Gag-protease PCR product via electroporation at 300 V and 500 ?F (27).
Following a 1-h incubation at room temperature, GXR cells were transferred to
T25 flasks containing 4 ml of medium each. Five days later, 5 ml R10 medium
was added to each flask. The percentage of infected cells was monitored from day
12 onwards by flow cytometry on a FACSCalibur flow cytometer (BD Bio-
sciences, San Jose, CA). Culture supernatants were harvested when approxi-
mately 30% of the GXR cells were infected and were stored in 1-ml aliquots at
?80°C for use in subsequent titration and replication assays.
Titration and replication assays. Titration of virus stocks and replication
assays were performed as previously described (2, 27, 38), using a multiplicity of
infection (MOI) of 0.003. The mean slope of exponential growth from days 3 to
6 was calculated using the semilog method in Excel. This was divided by the slope
of growth of the wild-type NL4-3 control included in each assay to generate a
normalized measure of replication capacity. Replication assays were performed
at least in duplicate, and results were averaged.
Sequencing of Gag-protease gene. The Gag-protease PCR product was diluted
1:15 in DEPC-treated water and population sequenced using Big Dye Termina-
tor ready reaction mix V3 (Applied Biosystems, Foster City, CA) and the fol-
lowing sequencing primers: 5? CTT GTC TAG GGC TTC CTT GGT 3? (nu-
cleotides 1098 to 1078), 5? CTT CAG ACA GGA ACA GAG GA 3? (nucleotides
991 to 1010), 5? GGT TCT CTC ATC TGG CCT GG 3? (nucleotides 1481 to
1462), 5? CAA CAA GGT TTC TGT CAT CC 3? (nucleotides 1755 to 1736), 5?
CCT TGC CAC AGT TGA AAC ATT T 3? (nucleotides 1981 to 1960), 5? TAG
AAG AAA TGA TGA CAG 3? (nucleotides 1817 to 1834), 5? CAG CCA AGC
TGA GTC AA 3? (nucleotides 2536 to 2520), and 5? GGA GCA GAT GAT
ACA GTA TT 3? (nucleotides 2331 to 2350). Sequences were analyzed on an
ABI 3130xl genetic analyzer (Applied Biosystems) and were visualized and
edited in Sequencher 4.8. Sequence data were aligned with the sequence of
HIV-1 subtype B reference strain HXB2 (GenBank accession no. K03455), using
a modified NAP algorithm (18), and insertions with respect to the HXB2 se-
quence were stripped from the sequences. A neighbor-joining tree was con-
structed from nucleotide sequences by using Paup 4.0 and was edited in Figtree
(http://tree.bio.ed.ac.uk/software/figtree/). Nucleotide differences between
plasma and recombinant virus Gag-protease sequences were quantified using
.html). BioEdit 7.0 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) was used to
calculate the similarity of each sequence to the consensus subtype C Gag se-
quence from 2004.
Identification of HLA-associated polymorphisms. Statistical methods (de-
scribed in detail in reference 5) that correct for phylogenetic relatedness between
HIV sequences, amino acid covariation, and HLA linkage disequilibrium effects
were used to identify HLA-associated polymorphisms. Briefly, a maximum like-
VOL. 84, 2010SUBTYPE C Gag AND HIV FITNESS10821
lihood phylogenetic tree was constructed for each gene, and a model of condi-
tional adaptation was inferred for each observed amino acid at each codon. In
this model, the amino acid is assumed to evolve independently down the phy-
logeny, until it reaches the observed sequences at the tree tips. In each host, the
selection pressure arising from HLA-restricted CTL and covariation between
HIV codons is modeled directly by a stochastic additive process. To identify
which factors contribute to the observed sequences, a forward selection proce-
dure is employed, in which the most significant association is iteratively added to
the model, with P values computed using the likelihood ratio test. Each observed
amino acid variant at each codon is evaluated in a binary fashion (presence
versus absence thereof). Multiple tests are addressed using q values, the P value
analogue of the false discovery rate (FDR), for each P value threshold (40). The
FDR is the expected proportion of false-positive results among results deemed
significant at a given threshold. For example, at a q value of ?0.2, we would
expect a false-positive proportion of 20% among identified associations.
Data analysis. Viral replication capacities were grouped according to the HLA
class I alleles expressed by the host. Analysis of variance (ANOVA) was used to
assess whether significant differences in replication capacities were observed
within expressed HLA-A, -B, and -C alleles. Then, for each individual allele (n ?
5), Student’s t test (or the Mann-Whitney U test in cases where the assumptions
of Student’s t test were not met) was used to compare replication capacities of
viruses generated from persons expressing versus not expressing the allele in
question. The relationships between replication capacity and log viral load, CD4
count, rate of CD4 decline, number of HLA-associated polymorphisms, and
sequence similarity to the HIV-1 subtype C Gag consensus were assessed using
Pearson’s correlation (for normally distributed variables) or Spearman’s rank
correlation (for non-normally distributed variables). Viruses were also catego-
rized according to the 10th and 90th percentiles of the replication capacity data,
into low- and high-replication-capacity groups, respectively. Clinical and se-
quence parameters were compared between these groups, using Student’s t test
(or the Mann-Whitney U test) or Fisher’s exact test (in the case of proportion
comparisons). The association between single amino acid residues in Gag-pro-
tease and the replication capacity was analyzed by Mann-Whitney U tests (uni-
variate method) and linear regression with a forward selection process (multi-
variate method). q values were calculated in both cases to account for multiple
comparisons (40). The significance cutoff for all analyses, unless indicated oth-
erwise, was a P value of ?0.05.
Nucleotide sequence accession numbers. Gag-protease sequences obtained in
this study are available in the GenBank database under accession numbers
HM593106 to HM593510.
Validation of generated Gag-protease NL4-3 recombinant
viruses. Gag-protease NL4-3 recombinant virus stocks from
406 subjects were generated in a median of 27 days (IQR, 23 to
32 days) following cotransfection of HIV-inducible, GFP-ex-
pressing T cells with Gag-protease-deleted NL4-3 plasmid and
clinically derived Gag-protease amplicons. To test whether re-
combinant viruses were representative of the original plasma
quasispecies, Gag-protease was resequenced from 40 randomly
selected recombinant viruses and compared with the original
plasma HIV RNA sequences. The median number of total
nucleotide differences between the recombinant virus se-
quence and the original plasma HIV RNA sequence (when
mixtures were not included as differences) was 0 (IQR, 0 to
1.5), resulting in an average nucleotide similarity of 0.99%
between pairs. The average number of nucleotide mixtures in
recombinant virus sequences was 21 (standard deviation
[SD] ? 17), indicating somewhat reduced diversity (Student’s
t test; P ? 0.0002) compared to that of the original plasma
sequences (mean ? 35; SD ? 36). Recombinant virus Gag
sequences closely clustered with respective plasma Gag se-
quences in a phylogenetic tree (see Fig. SA1 in the supplemen-
tal material). These data indicate that the Gag-protease re-
combinant viruses were representative of the original plasma
quasispecies. All further analyses were based on the original
plasma HIV sequences.
Assay variability. The replication capacities of Gag-protease
NL4-3 recombinant viruses were assayed in duplicate, inde-
pendently, in an HIV-1-inducible GFP reporter cell line. Rep-
lication capacity was defined as the slope of the increase in the
percentage of infected cells from days 3 to 6 following infec-
tion, normalized to wild-type NL4-3. Duplicate measurements
were highly concordant (Pearson’s correlation; r ? 0.88 and
P ? 0.0001). Accuracy of recombinant viral titers was achieved:
on day 3 of the assay, the mean % GFP-expressing cells was
0.65% (SD ? 0.28%). Importantly, the observed variability in
day 3 readings did not influence viral replication capacity mea-
surements (Pearson’s correlation; r ? 0.04 and P ? 0.44).
Distribution of replication capacities of recombinant vi-
ruses. The NL4-3-normalized replication capacities of the re-
combinant viruses generated from the 406 cohort participants
approximated a normal distribution (mean ? 0.62; SD ? 0.1)
(Fig. 1). The replication capacities of the recombinant HIV-1
subtype C Gag-protease sequences inserted into the NL4-3
backbone were considerably lower than those of the wild-type
NL4-3 control and 25 subtype B Gag-protease NL4-3 recom-
binant viruses, whose mean replication capacity normalized to
wild-type NL4-3 was 0.95 (SD ? 0.13) (data not shown).
Association of recombinant virus replication capacities with
specific HLA types. Replication capacities of recombinant vi-
ruses were grouped according to HLA alleles expressed by the
host (Fig. 2). Overall, replication capacities varied significantly
between the different HLA-B alleles (ANOVA; P ? 0.01) but
not between HLA-A or HLA-C alleles, suggesting that HLA-B
alleles have the greatest impact on Gag-protease-mediated
replication capacity. Relationships between specific HLA al-
leles and replication capacity were also observed, the strongest
of which was the association of HLA-B*81 with lower replica-
tion capacities (Student’s t test; P ? 0.0001). P values pre-
sented are uncorrected for multiple comparisons. Only the
association of HLA-B*81 with lower replication capacities
FIG. 1. Distribution of Gag-protease NL4-3 recombinant virus rep-
lication capacities, normalized to the growth of wild-type NL4-3.
10822WRIGHT ET AL. J. VIROL.
would remain statistically significant following Bonferroni ad-
justment for multiple comparisons.
Besides HLA-B*81, other alleles that were associated with
low-replication-capacity recombinant viruses were HLA-
B*5801, HLA-A*0205 (Mann-Whitney U test; P ? 0.05 and
P ? 0.04, respectively), HLA-A*3009, and HLA-A*3001 (Stu-
dent’s t test; P ? 0.02 and P ? 0.05, respectively). Due to tight
linkage between HLA-B*5801 and HLA-A*0205 (D? ? 0.56
), the allele driving the effect could not be identified.
Among 5 individuals with HLA-B*3009, 4 possessed HLA-
B*81, which likely explains the association of HLA-B*3009
with lower replication capacities.
Alleles associated with higher replication capacities were
HLA-Cw*0702 (Student’s t test; P ? 0.05) and HLA-Cw*0501
(Mann-Whitney U test; P ? 0.001). Only 6 individuals in this
study possessed HLA-Cw*0501, and all were linked to repli-
cation capacities above the 80th percentile of the data set
(?0.7). HLA-Cw*0501 is in linkage disequilibrium with HLA-
B*1801—5 of 6 individuals with HLA-Cw*0501 also carried
HLA-B*1801—and could therefore partly contribute to the
disadvantage associated with HLA-B*1801 in subtype C infec-
In an additional analysis, the HLA types of the individuals
corresponding to the fittest recombinant viruses (?90th per-
centile of the data set, i.e., ?0.74; n ? 41) were compared to
the HLA types of individuals with the least-fit recombinant
viruses (10th percentile of the data set, i.e., ?0.5; n ? 41).
Protective alleles were defined as those that were most strongly
associated with lower viral loads in HIV-1 subtype C-infected
individuals, namely, HLA-B*57, HLA-B*5801, and HLA-
FIG. 2. Associations between HLA alleles and replication capacities of Gag-protease NL4-3 recombinant viruses. (A to C) Graphs show the
mean (dot), median (vertical line), interquartile range (edges of boxes), and most extreme values (edges of whiskers) of replication capacities for
each different HLA allele for which n was ?5.*, individual significant (P ? 0.05) associations (Student’s t test);**, associations that survive
Bonferroni correction for multiple comparisons. Overall P values (ANOVA) indicate the significance of differences in replication capacity between
alleles of each group. (D) Graph showing a significantly greater proportion (Fisher’s exact test) of individuals with protective HLA alleles
(HLA-B*57, HLA-B*5801, and HLA-B*81) in the group with the least-fit viruses (replication capacity of ?0.5; n ? 41) than in the group with the
fittest viruses (replication capacity of ?0.74; n ? 41).
VOL. 84, 2010SUBTYPE C Gag AND HIV FITNESS10823
B*8101 (20), and were also found later to be the most strongly
associated with lower viral loads or higher CD4 counts in a
cohort of over 1,000 HIV-1 subtype C-infected individuals
(43). The proportion of individuals possessing a protective
allele was significantly greater in the low-replication-capacity
group than in the high-replication-capacity group (Fisher’s ex-
act test; P ? 0.003) (Fig. 2D).
When HLA-A, -B, and -C alleles were ranked according to
viral load and then according to replication capacity, the ranks
correlated positively with one another for each group of HLA
alleles, although not significantly for HLA-C alleles (Spear-
man’s rank correlation; r ? 0.43 and P ? 0.03, r ? 0.42 and P ?
0.04, and r ? 0.47 and P ? 0.06, respectively), which indicates
a relationship between viral load and Gag-protease replication
capacity (data not shown).
Correlation between replication capacity and baseline log
viral load or CD4 count. Replication capacities of recombinant
viruses correlated positively with baseline log viral loads
(Spearman’s rank correlation; r ? 0.24 and P ? 0.0001) and
negatively with baseline CD4 counts (Spearman’s rank corre-
lation; r ? ?0.17 and P ? 0.0004) (Fig. 3A and B). These
effects remained after removal of the protective alleles HLA-
B*57, HLA-B*5801, and HLA-B*81 from analysis (Spear-
man’s rank correlation; r ? 0.18 and P ? 0.001 for baseline log
viral load and r ? ?0.14 and P ? 0.01 for baseline CD4 count).
Interestingly, analysis of the relationship between viral load or
CD4 count and replication capacity among individuals express-
ing these protective alleles also revealed a significant positive
correlation (Pearson’s correlation; r ? 0.33 and P ? 0.001 for
viral load and r ? ?0.33 and P ? 0.001 for CD4 count).
Relationship between replication capacity and subsequent
longitudinal rate of CD4 decline. An average of 2.28 years
(SD ? 1.3 years) of untreated follow-up was available for 339
Sinikithemba patients. For each study subject, linear regression
was used to compute the rate of CD4 decline for the duration
of untreated clinical follow-up. Spearman’s correlation was
then used to investigate the relationship between viral replica-
tion capacity and subsequent rate of CD4 decline. We ob-
served no statistically significant relationship overall between
replication capacity and CD4 decline (Spearman’s rank corre-
lation; r ? ?0.01 and P ? 0.79). Stratification of the analysis by
baseline CD4 counts (?200, ?200, ?350, and ?350 cells/mm3)
also failed to reveal any significant correlations between rep-
lication capacity and the rate of CD4 decline (not shown).
Figure 3C shows a lack of correlation between CD4 decline
and Gag-protease-mediated replication capacity at baseline
CD4 counts of ?200 cells/mm3(Spearman’s rank correlation;
r ? ?0.02 and P ? 0.73).
Association of sequence variability in Gag with replication
capacity. (i) Overall variability. To investigate whether an
increasing number of polymorphisms in Gag would tend to
reduce replication capacity, the percent amino acid similarities
of Gag sequences to the 2004 consensus subtype C Gag se-
quence were calculated using the sequence identity matrix
function in BioEdit 7.0 and correlated with replication capac-
ity. Unexpectedly, the calculated Gag percent similarity corre-
lated negatively, although weakly, with replication capacity
(Pearson’s correlation; r ? ?0.18 and P ? 0.0004), i.e., the
fittest viruses were generally least like the consensus sequence
(Fig. 4A). This analysis was repeated separately for each region
of Gag, namely, p17, p24, p7, and p6, to see whether this
relationship differed between regions. There remained an in-
verse relationship between percent similarity to consensus and
replication capacity for every region of Gag except p24, al-
though this was statistically significant only for p17 and p7 (Fig.
4B). There was no correlation between percent similarity to
the subtype C Gag p24 consensus and replication capacity. In
contrast, the majority of nonconsensus residues in p17/p7 in-
creased replication capacity. It should be noted that divergence
from the consensus subtype C sequence did not represent
convergence to the consensus subtype B sequence, which
would have indicated that divergence from the consensus sub-
type C sequence resulted in better compatibility with the sub-
type B NL4-3 backbone, and therefore in fitter viruses.
(ii) HLA-associated variability. HLA-associated polymor-
phisms—amino acids that are significantly more likely to occur
FIG. 3. Correlations between replication capacities of recombinant
viruses encoding patient Gag-protease and markers of disease progres-
sion. The graphs show replication capacity versus baseline log viral
load (A), baseline CD4 count (B), and rate of CD4 decline (C).
Correlations were calculated with Spearman’s rank correlation test.
10824WRIGHT ET AL.J. VIROL.
FIG. 4. Associations between sequence variability in Gag-protease and replication capacities of recombinant viruses encoding Gag-protease.
(A) Significant negative correlation (Pearson’s correlation; n ? 405) between percent similarity of Gag sequences to the consensus subtype C Gag
sequence and replication capacity. (B) Gag p17, p24, p6, and p7 percent similarity to consensus subtype C sequence versus replication capacity
(Spearman’s rank correlation; n ? 405). (C) Significant positive correlation (Pearson’s correlation; n ? 17) between entropy of HLA-associated
sites in or within five amino acids of Gag epitopes and the average replication capacity of viruses with mutations at these sites. (D and E) Significant
differences (Fisher’s exact test) in proportions of p24 variant epitopes and p24 consensus epitopes (D) and of TL9 variant epitopes and TL9
consensus epitopes (E) between the least-fit viruses (replication capacity of ?0.5; n ? 41) and the fittest viruses (replication capacity of ?0.74; n ?
41). (F and G) Significant differences (Fisher’s exact test) in proportions of nonconsensus amino acids associated with decreased replication
capacity (RC) in p24 versus p17 (n ? 12 and n ? 23) (F) and in HLA-A- versus HLA-B-restricted epitopes (n ? 13 and n ? 17) (G).
VOL. 84, 2010 SUBTYPE C Gag AND HIV FITNESS 10825
in the presence of a particular HLA allele—were identified in
the current data set by use of methods that take into account
the phylogenetic relatedness of sequences, amino acid covaria-
tion, and HLA linkage disequilibrium effects (5). Each se-
quence was then analyzed in the context of the patient’s HLA
class I profile, and the number of HLA-associated polymor-
phisms was computed. To further analyze the influence of
HLA alleles on Gag-protease replication capacity, the com-
puted polymorphisms were correlated with replication capac-
ity. The numbers of HLA-A-, -B-, and -C-associated polymor-
phisms in each sequence did not correlate significantly with
replication capacity overall. Likewise, no dose-dependent ef-
fects of polymorphisms on replication capacity were observed
among polymorphisms associated with protective HLA types.
Similarly, when the relationship between the number of HLA-
associated polymorphisms and replication capacity was inves-
tigated irrespective of patient HLA class I profile, i.e., also
taking into account inherited polymorphisms, no significant
associations were found. Therefore, while some HLA-associ-
ated polymorphisms significantly impact replication capacity
(8, 45), the sum of HLA-selected polymorphisms, irrespective
of location in Gag, was not associated with replication capacity
in this chronic infection cohort. There was, however, a weak
trend (Spearman’s rank correlation; r ? ?0.09 and P ? 0.08)
toward lower replication capacities with increasing numbers of
HLA-associated polymorphisms in epitopes or within five
amino acids of epitopes restricted by the selecting HLA allele
(these polymorphisms are more likely to represent escape mu-
tations, not secondarily arising compensatory mutations ).
Previously, increasing numbers of HLA-B-associated polymor-
phisms in or within five amino acids of Gag epitopes were
strongly associated with lower viral loads in early infection, and
this was attributed to lower fitness levels of these viruses (15).
The number of HLA-B-associated polymorphisms in or within
five amino acids of Gag epitopes was negatively correlated with
fitness (Spearman’s rank correlation; r ? ?0.11 and P ? 0.03),
although not strongly so. The relatively weak relationship be-
tween the number of HLA-associated polymorphisms in Gag
and replication capacity in the present chronic infection cohort
might be explained by the accumulation of compensatory mu-
tations during the course of infection. In fact, evidence has
been found for a strong effect of HLA-mediated selection
pressure in Gag on replication capacity in early infection and
no such significant relationship in the very late chronic stage of
infection, suggesting that this effect wanes over time, presum-
ably due to the development of compensatory mutations
(M. A. Brockman et al., submitted for publication).
Since there is some evidence that HLA-associated escape
mutations occurring in conserved sites of HIV carry a greater
fitness cost than those occurring in regions of high variability
(45), we compared the average replication capacity of viruses
possessing each HLA-associated polymorphism with the cor-
responding entropy at that position. A trend toward a signifi-
cant correlation between these two parameters was found
(Pearson’s correlation; r ? 0.24 and P ? 0.06). When the
analysis was restricted to those polymorphisms in epitopes or
within five amino acids of epitopes restricted by the selecting
HLA allele, the correlation was much stronger (Pearson’s cor-
relation; r ? 0.68 and P ? 0.003) (Fig. 4C). Thus, HLA-
associated escape mutations at more conserved sites (with
lower entropy) in Gag were associated with greater fitness
(iii) Epitope variability: association of TL9 variant with
lower replication capacity. Next, the relationship between se-
quence variability in specific HLA-restricted epitopes and rep-
lication capacity was examined. The proportion of variant Gag
epitopes (i.e., nonconsensus) versus consensus epitopes was
compared between the least-fit and fittest virus groups by Fish-
er’s exact test, and no significant difference was found. How-
ever, there were marginally more variant Gag p24 epitopes in
the sequences from the least-fit group (Fisher’s exact test; P ?
0.04) (Fig. 4D). This significant result was driven mainly by the
greater proportion of variant HLA-B*81-restricted TL9
epitopes in the least-fit group (Fisher’s exact test; P ? 0.007)
(Fig. 4E), although there were also significantly more variant
HLA-B*57-restricted QW9 epitopes in viruses of lower fitness
(Fisher’s exact test; P ? 0.04) (data not shown).
(iv) Single amino acid associations with replication capac-
ity. In an exploratory analysis, the Mann-Whitney U test was
used to identify specific amino acids in Gag-protease associ-
ated with increased or decreased replication capacity. Al-
though none of the comparisons yielded Q values of ?0.2, 58
associations with P values of ?0.05 were found for Gag, and 9
were found for protease, when consensus-nonconsensus pairs
were counted as a single association (n ? 5 for both groups
compared [see Table SA1 in the supplemental material]). Of
the 58 associations in Gag, 23 occurred in p17, 12 in p24, 3 in
the p2 linker peptide, 9 in p7, and 11 in p6.
Considering amino acids in Gag associated with alterations
in viral replication capacity, most of the nonconsensus amino
acids in p24 were associated with lower replication capacity
(10/12 residues), while most of the nonconsensus residues in
p17 were associated with increased replication capacity (15/23
residues). This difference was statistically significant (Fisher’s
exact test; P ? 0.01) (Fig. 4F). Only 17 of 58 amino acids
associated with replication capacity alterations corresponded
to an HLA association at that position (not necessarily with the
same amino acid), and 11 of these were HLA-B associated.
Twenty-six associations occurred in published or previously
predicted epitopes (13, 36), with 13 in HLA-A-restricted
epitopes, 17 in HLA-B-restricted epitopes, and 6 in HLA-C-
restricted epitopes (10 of these occurred in epitopes that were
restricted by more than one HLA allele class and were thus
considered under more than one category). Within HLA-B-
restricted epitopes, 13 (8 of these were in p24) of 17 noncon-
sensus amino acids were associated with decreased replication
capacity, while in the HLA-A-restricted epitopes, 8 of 13 non-
consensus amino acids (10 of these were in p17) were linked
with increased replication capacity (Fisher’s exact test; P ?
0.06) (Fig. 4G). These results are suggestive of HLA-B-medi-
ated selective pressure on Gag p24, with resulting lower rep-
lication capacity. It should also be noted that half of the amino
acids associated with changes in Gag-protease-mediated rep-
lication capacity were neither HLA class I associated nor
within known or predicted epitopes.
Multivariate analysis (linear regression with forward selec-
tion) was also undertaken. Seventeen of the 58 associations in
Gag and 4 of the 9 associations in protease identified by uni-
variate analysis also had P values of ?0.05 (but Q values of
?0.2) in the multivariate model, and the strongest of these
10826WRIGHT ET AL. J. VIROL.
associations was the consensus T at position 186, with in-
creased replication capacity (see Table SA1 in the supplemen-
(v) HLA-B*81-associated T186S mutation is linked to lower
replication capacity. Since the consensus residue 186T was
strongly associated with an increased replication capacity,
changes away from consensus at this position were compared
between the least-fit and fittest viruses. In the least-fit group, 9
sequences had an S and 1 had an A at position 186, while only
1 had an S at this position in the fittest group (Fisher’s exact
test; P ? 0.01) (Fig. 5A). With the exception of 1 sequence with
186A and another sequence which had a mixture of T and S at
this position, the nonconsensus amino acid at codon 186 was S.
Overall, 186S was associated with a decrease in replication
capacity compared with that for the 186T consensus (Student’s
t test; P ? 0.006; n ? 403) (Fig. 5B). This polymorphism is
associated with HLA-B*81 and occurs in the HLA-B*81-
restricted epitope TL9. The difference in numbers of variant
TL9 epitopes between the low- and high-fitness groups could
be attributed largely to variability at position 186. However,
when only HLA-B*81-positive individuals were considered,
the replication capacities of viruses with 186S and 186T were
both below average and were not significantly different from
one another (data not shown), indicating that other mutations
are also responsible for the lower fitness of viruses from these
individuals. The lack of difference in replication capacity be-
tween viruses with 186S and 186T from individuals with HLA-
B*81 may also suggest that the fitness cost of 186S was com-
pensated for in some cases.
(vi) Residues covarying with 186S. Codon covariation lists
were generated from the current data set as previously de-
scribed (5). Amino acids positively associated with 186S and/or
negatively associated with 186T included 177D, 182S, 190A,
190I, 256I, and 343I (P ? 0.05; Q ? 0.2). Amino acids nega-
tively associated with 186S and/or positively associated with
186T included 65Q, 177E, 190T, 256V, and 343L (P ? 0.05;
Q ? 0.2). Replication capacities of viruses with 186S and var-
ious numbers of associated residues (Q65X, E177X, Q182S,
T190X, V256X, and L343X) were compared to assess whether
these might function as compensatory mutations. The number
of covarying residues present correlated positively but not sig-
nificantly with replication capacity (Pearson’s correlation; r ?
0.26 and P ? 0.19). However, on closer examination of se-
quences with 186S, a greater occurrence of mutations at posi-
tions 182 and 190 (but not at other covarying positions) was
noted in the fitter viruses (Fig. 5C). This was statistically sig-
nificant (Student’s t test; P ? 0.006), suggesting that 190X and
182S, which occur parallel to and on either side of residue 186
in a helix structure, might indeed be compensatory mutations.
The mechanisms underlying HIV-1 control by protective
HLA alleles are not fully understood and could involve target-
ing of functionally important epitopes in Gag, resulting in
selection of escape mutations with a fitness cost. Therefore,
this study was undertaken to investigate, at the population
level, the impact of HLA-mediated immune pressure in Gag
on viral fitness and its impact on HIV-1 pathogenesis.
Our results showed an association between protective HLA
alleles (HLA-B*57, HLA-B*5801, and HLA-B*81) and lower
Gag-protease replication capacities. Since (i) protective HLA
alleles were associated with lower viral loads, (ii) Gag-protease
replication capacity correlated with viral loads even on removal
of protective HLA alleles from the analysis and within individ-
uals with protective alleles, and (iii) replication capacity
ranked according to HLA-A, -B, and -C alleles correlated
significantly with the ranks according to viral load, the possi-
FIG. 5. Single Gag amino acid associations with altered replication
capacities of Gag-protease NL4-3 recombinant viruses. (A) Greater
proportion (Fisher’s exact test) of 186S/A residues in the least-fit
viruses (replication capacity of ?0.5; n ? 41) than in the fittest viruses
(replication capacity of ?0.742; n ? 41). (B) Lower replication capac-
ity of viruses with 186S (n ? 27) than that of viruses with the consensus
186T (n ? 376; Student’s t test). (C) Significantly higher replication
capacities of viruses with 186T (n ? 376) and with 186S with putative
compensatory mutations 190X and 182S (n ? 18) than those of viruses
with 186S alone (n ? 9; Student’s t test).
VOL. 84, 2010 SUBTYPE C Gag AND HIV FITNESS10827
bility that HLA alleles and replication capacity are indirectly
related to each other through association with viral load cannot
be excluded. However, since mutations in Gag selected by the
protective HLA alleles B*5703 and B*5801 were shown to
significantly decrease the overall replication capacity of iso-
lates and to confer benefits on infant and adult recipients (7, 8,
15, 33, 39), except in the presence of compensatory mutations
(39), it seems very likely that a direct relationship exists be-
tween HLA alleles and Gag-protease replication capacity.
Gag-protease replication capacity varied significantly between
the different HLA-B but not HLA-A or HLA-C alleles, con-
sistent with the idea that HLA alleles influence Gag-protease
replication capacity through selecting mutations, as HLA-B
alleles have the greatest selection pressure (20). Moreover,
increasing numbers of HLA-B-associated mutations in or
flanking epitopes (likely HLA-selected escape mutations) cor-
related with decreased HIV replication capacities. In further
support of a direct relationship between protective HLA al-
leles and replication capacity, HLA-B*81 was by far the allele
most strongly associated with lower replication capacity, even
though HLA-B*5703-positive individuals had a lower average
viral load than HLA-B*81-positive individuals, and 186S
present in the HLA-B*81-restricted epitope TL9 (positions
180 to 188) was the mutation most strongly associated with
lowered replication capacity, thereby providing a possible
mechanism for the influence of HLA-B*81 on replication ca-
pacity. TL9 was previously described as one of the key Gag
epitopes under strong selection pressure by a beneficial HLA
allele, with variance mainly at residues 182 and 186 (both with
changes predominantly to serine) (14). Interestingly, in a re-
cent study, the number of public T-cell clonotypes specific for
simian immunodeficiency virus (SIV) Gag CM9 (residues 181
to 189), which occurs in nearly the exact same position as TL9
in HIV, correlated strongly and negatively (r2? ?0.71) with
the viral set point in rhesus macaques (34). Residue 186 in
HIV Gag has also been classified as a site where mutations
revert upon transmission to a host lacking the HLA allele that
selected them, presumably due to a fitness cost (26). It should
be noted, though, that differences in fitness associated with
variability at position 186 did not translate into viral load
differences in this chronic infection cohort (data not shown),
which could suggest that the fitness cost of the 186S mutation
may be compensated in some cases, and therefore not of last-
ing benefit, and that the balance between the fitness cost of
186S and an effective CTL response to TL9 may be important
in determining the outcome. However, taking the results to-
gether, it seems likely that protective HLA alleles, in particular
HLA-B*81, influence Gag-protease
through CTL selection pressure and that this may partly con-
tribute to their protective effect. From the present data, this
seems likely to be a more prominent mechanism of protection
for HLA-B*81 than for HLA-B*57 and HLA-B*5801 in sub-
type C infection.
Given our observation that lower Gag-protease replication
capacities were related to protective HLA types, lower baseline
viral loads, and higher baseline CD4 counts, we wished to
investigate whether viral replication capacity may also corre-
late with the subsequent rate of CD4 decline during chronic
infection. However, such a correlation was not observed in the
present study. This may be explained partly by the balance that
exists between Gag CTL responses and replication capacity in
influencing clinical outcomes. Accumulation of escape muta-
tions in HIV carries a fitness cost to the virus, but the disad-
vantage to the virus is offset by the advantage of escaping
effective CTL responses that were holding replication in check,
resulting in increased viral loads and accelerated disease pro-
gression despite a replication-deficient virus (8, 19). Another
consideration is that replication capacity is not static and com-
pensatory mutations may have developed at a time point later
than that measured, influencing the subsequent rate of CD4
decline. Data from the present study and previous studies
suggest that mutations with a fitness cost are readily compen-
sated. The T186S mutation was most strongly associated with
decreased replication capacity, yet in the presence of covarying
mutations at positions 182 and 190, the mean replication ca-
pacity was not significantly different from the mean for the
entire cohort, suggesting that the possible fitness cost of this
mutation was compensated in these cases. Therefore, although
there may be a benefit to decreased replication capacity (as
supported by cross-sectional correlations with viral loads and
CD4 counts), the data do not support an enduring benefit or a
lasting significant impact of Gag-protease replication capacity
on the rate of disease progression, at least once the chronic
infection stage has been reached. The results of Brockman et
al. (submitted) are consistent with this notion. However, acute
infection studies and/or longitudinal analysis of replication ca-
pacity and sequence changes, together with CTL responses,
may be necessary to better assess the relative impact of each on
disease progression. Site-directed mutagenesis experiments
would also be necessary to confirm the suspected fitness costs
and compensatory roles of some of the mutations described
The data support the hypothesis that mutations at conserved
residues/regions, in particular in conserved Gag p24 as op-
posed to the less-conserved Gag p17, are more likely to result
in a fitness cost: HLA-associated escape mutations at con-
served sites were associated with lower replication capacities,
there were significantly more variant p24 epitopes in the least-
fit viruses than in the fittest viruses, and most of the mutations
significantly associated with altered replication capacities in
p24 decreased replication capacity, while most in p17 increased
replication capacity. In agreement with these data, beneficial
HLA alleles in an African cohort were associated with strong
selection at key epitopes which occurred mostly in Gag p24
(14), and there is recent evidence that HLA-B*57 mediates its
protective effect mainly through attenuating mutations in Gag
p24 (39). Furthermore, the breadth of Gag p24, but not p17 or
p15, CD8 T-cell responses in HLA-B*13-positive individuals
was significantly associated with decreasing viral loads (17).
Taken together, the data generally support the inclusion of
conserved regions such as Gag p24 in a vaccine that is aimed at
driving HIV toward a less-fit state.
Interestingly, a larger number of amino acid differences
from the consensus subtype C Gag sequence were weakly
but significantly associated with increasing viral fitness. The
percent amino acid similarity to the consensus subtype C
Gag sequence also correlated negatively with viral load and
positively with CD4 count (data not shown), suggesting that
more changes from consensus and increased fitness of vi-
ruses may occur with disease progression. In fact, the fitness
10828WRIGHT ET AL.J. VIROL.
of HIV isolates was previously shown to increase with dis-
ease progression (44). Consensus amino acids could, in
some instances, be escape mutations in response to common
HLA alleles, but we speculate that they represent the non-
escape form in the majority of cases and that nonconsensus
residues represent escape and compensatory mutations in
response to CTL and non-CTL immune pressure, although
they could also represent random mutations. Based on this
conjecture, we suggest that more changes away from con-
sensus likely indicate more compensation, and therefore
fitter viruses. Another explanation is that the majority of
mutations introduced into HIV are likely to have no or little
fitness cost or to actually increase fitness. Consistent with
this idea, p17 and p7 were significantly more divergent from
the consensus than p24 was, i.e., significantly more muta-
tions occurred in p17 and p7 than in p24, and the percent
similarity to consensus for both p17 and p7 was negatively
correlated overall with fitness, while there was no correla-
tion for p24. The direct relationship between replication
capacity and the entropy of mutated sites in the present
study, as well as the recent finding that escape mutations in
conserved Gag p24 carry significant fitness costs while most
of the escape mutations in the highly variable env gene are
fitness neutral or increase fitness (45), lends further support
to this argument.
Another interesting finding was that most of the muta-
tions in Gag associated with altered replication capacity
were not HLA associated (71%). It should be noted, how-
ever, that a limitation of this study was the insertion of
subtype C Gag-protease into a subtype B backbone, and
therefore some Gag-protease mutations associated with al-
tered replication capacity might represent those that inter-
act with other components of the backbone. A significantly
lower replication capacity of subtype C/B recombinants than
that of subtype B recombinants was observed, which could
suggest that mixing of subtypes results in suboptimal repli-
cation. Alternatively, this finding could mean that Gag-pro-
tease function is inferior in subtype C versus subtype B
viruses, which may partly explain previously described fit-
ness differences between subtypes (1). Further experiments
are required to discriminate between these possibilities.
Supporting the latter rather than the former possibility,
convergence of subtype C Gag sequences to the consensus
subtype B sequence was not associated with fitter recombi-
nant viruses. Furthermore, the findings of the present study
are in agreement with those of Brockman et al. (submitted),
which show that subtype B Gag-protease NL4-3 recombi-
nant viruses correlate with cross-sectional viral load and
CD4 count data as well as with specific HLA types, strongly
supporting the hypothesis that the current assay system is
clinically and biologically relevant.
In summary, there is evidence that protective HLA al-
leles, especially HLA-B*81, influence subtype C HIV repli-
cation capacity through selection of mutations in Gag that
incur a fitness cost. Moreover, mutations in conserved
rather than more-variable regions of Gag are more likely to
carry a fitness cost, suggesting that conserved regions such
as Gag p24 should be included in a vaccine aiming to drive
HIV toward a less-fit state. However, the long-term clinical
impact of immune-driven fitness costs requires further in-
vestigation, given the evidence for compensation and the
observation that replication capacity does not correlate with
the subsequent rate of CD4 decline in chronic infection.
This research was funded by the NIH (grant ROI-AI067073, con-
tract NOI-AI-15422), the South African AIDS Vaccine Initiative, and
the Ragon Institute Fund for Innovation and New International Initi-
atives. J.K.W. was funded by the National Research Foundation and
the Ragon Institute of Massachusetts General Hospital, Massachusetts
Institute of Technology, and Harvard University. Z.L.B. was supported
by a New Investigator Award from the Canadian Institutes for Health
Research (CIHR). T.N. holds the South African Department of Sci-
ence and Technology/National Research Foundation Research Chair
in Systems Biology of HIV/AIDS.
We thank Jennifer Sela, Pamela Rosato, and Taryn Green for tech-
nical assistance; Johannes Viljoen and the Africa Centre laboratory for
providing access to tissue culture and sequencing facilities; the Durban
clinic staff (Sisters Kesia Ngwenya, Thandi Cele, Thandi Sikhakane,
and Nokuthula Lutuli); and Isobel Honeyborne, Wendy Mphatswe,
and the management of McCord Hospital for their support of the
Sinikithemba cohort. Finally, we thank and acknowledge the Sini-
kithemba cohort study participants.
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