JOURNAL OF VIROLOGY, Apr. 2006, p. 3617–3623
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 7
Fitness Cost of Escape Mutations in p24 Gag in Association with
Control of Human Immunodeficiency Virus Type 1
Javier Martinez-Picado,1Julia G. Prado,1Elizabeth E. Fry,2Katja Pfafferott,3Alasdair Leslie,3
Senica Chetty,4Christina Thobakgale,4Isobel Honeyborne,3Hayley Crawford,3
Philippa Matthews,3Tilly Pillay,3Christine Rousseau,5James I. Mullins,5
Christian Brander,6Bruce D. Walker,6David I. Stuart,2
Photini Kiepiela,4and Philip Goulder3,6*
irsiCaixa Foundation, University Hospital “Germans Trias i Pujol,” Barcelona, Spain1; Division of Structural Biology,
Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN,
United Kingdom2; Department of Paediatrics, Nuffield Department of Medicine, Peter Medawar Building for
Pathogen Research, South Parks Road, Oxford OX1 3SY, United Kingdom3; HIV Pathogenesis Programme,
The Doris Duke Medical Research Institute, University of Natal, Durban, South Africa4;
Department of Microbiology, University of Washington, Seattle, Washington 981955;
and Partners AIDS Research Center, Massachusetts General Hospital,
13th Street, Building 149, Charlestown, Boston, Massachusetts 021296
Received 15 October 2005/Accepted 9 December 2005
Mutational escape by human immunodeficiency virus (HIV) from cytotoxic T-lymphocyte (CTL) recognition
is a major challenge for vaccine design. However, recent studies suggest that CTL escape may carry a sufficient
cost to viral replicative capacity to facilitate subsequent immune control of a now attenuated virus. In order
to examine how limitations can be imposed on viral escape, the epitope TSTLQEQIGW (TW10 [Gag residues
240 to 249]), presented by two HLA alleles associated with effective control of HIV, HLA-B*57 and -B*5801, was
investigated. The in vitro experiments described here demonstrate that the dominant TW10 escape mutation,
T242N, reduces viral replicative capacity. Structural analysis reveals that T242 plays a critical role in defining
the start point and in stabilizing helix 6 within p24 Gag, ensuring that escape occurs at a significant cost. A
very similar role is played by Thr-180, which is also an escape residue, but within a second p24 Gag epitope
associated with immune control. Analysis of HIV type 1 gag in 206 B*57/5801-positive subjects reveals three
principle alternative TW10-associated variants, and each is strongly linked to concomitant additional variants
within p24 Gag, suggesting that functional constraints operate against their occurrence alone. The extreme
conservation of p24 Gag and the predictable nature of escape variation resulting from these tight functional
constraints indicate that p24 Gag may be a critical immunogen in vaccine design and suggest novel vaccination
strategies to limit viral escape options from such epitopes.
Virus-specific cytotoxic T-lymphocyte (CTL) activity plays a
central role in the control of immunodeficiency virus infection
(18). Initial studies suggested that mutations resulting in a
reduction of viral recognition by CTL—CTL escape—lead to
diminished immune control (29). In certain instances, muta-
tional escape has appeared to precipitate a rapid progression
to AIDS (4, 19, 23). More recently, however, examples of CTL
escape have been described for acute infections associated with
subsequent successful control of viremia (11, 24, 26). In each
case (22), and also in an example of escape occurring in
chronic infection (12), reversion to the wild type was observed
when the relevant viral mutant was transmitted to major his-
tocompatibility complex-mismatched recipients. Reversion in
the absence of the CTL-mediated pressure driving the selec-
tion of the escape mutant suggests a cost to viral replicative
capacity incurred by acquisition of the relevant escape muta-
tion. This was confirmed for examples from the simian immu-
nodeficiency virus (SIV) macaque model (5, 11, 12, 13, 26) but
has yet to be demonstrated for human immunodeficiency virus
type 1 (HIV-1) infection.
All examples published to date of reversion posttransmission
are in epitopes within the capsid protein. Pressure for CTL
escape in this conserved protein (34) is likely to be strongly
opposed by selection pressure to resist amino acid sequence
change. To better understand the mechanisms underlying ef-
fective control of HIV infection, we investigate here, in more
detail, escape occurring within the p24 Gag epitope TSTLQE
QIGW (TW10 [Gag residues 240 to 249]) that dominates the
acute CTL response in individuals expressing HLA-B*57 or
-B*5801 (2). These are the two HLA class I molecules inde-
pendently associated with control of HIV-1 infection in South
Africa (21). The commonest escape mutation, Thr3Asn at
Gag-242 (T242N), reverts to Thr-242 following transmission
to HLA-B*57/5801-negative individuals (24). The structural
role of T242 within the capsid protein is described, and the
consequences of the T242N mutation are evaluated. Three
additional TW10 mutational escape options for HIV-1 are
identified. In each case, the coexistence of additional, puta-
tive compensatory mutations is observed, indicating the ex-
treme limitations on escape from this p24 Gag-specific
* Corresponding author. Mailing address: Peter Medawar Building,
South Parks Rd., Oxford OX1 3SY, United Kingdom. Phone: 44-1865-
281884. Fax: 44-1865-281236. E-mail: firstname.lastname@example.org.
MATERIALS AND METHODS
Study cohorts. The study cohorts used here have been previously described
(24). The 258 C-clade-infected subjects analyzed in this study were collected
from Durban, South Africa, and predominantly consisted of Zulu/Xhosa women
recruited from the Cato Manor and St. Mary’s Hospital, Mariannhill, South
Africa, antenatal clinics. These subjects were all antiretroviral therapy (ART)
naı ¨ve. The median viral load was 21,900 HIV RNA copies/ml plasma (range, ?50
to 6.88 ? 106), and the median absolute CD4 count was 449/mm3(range, 101 to
1,215/mm3). The 187 B-clade-infected subjects were collected from diverse
sources encompassing Europe, the Caribbean, and North America. Seventeen
percent of these subjects were receiving ART at the time of analysis. The median
viral load in those not receiving ART was 24,600 RNA copies/ml plasma (range,
?400 to ?750,000). The median CD4 count for those not receiving ART was
420/mm3(range, 131 to 1,280/mm3).
Construction of p24 recombinant virus. Proviral DNA was extracted from
peripheral blood mononuclear cells from an HLA-B57-negative child (SMH-05-
Child) who had been vertically infected from his HLA-B57-positive mother (24).
The sample corresponded to a sample with a mixture of Thr and Asn at Gag
residue 242 (24). The p24 capsid coding region of gag was PCR amplified using
the primers 5?-GAT AGA GGA AGA GCA AAA CAA A-3? (positions 1098 to
1119 in HIV-1HXB2; a SapI restriction site is underlined) and 5?-TTT TTC CTA
GGG GCC CT-3? (positions 2005 to 2021; an ApaI restriction site is underlined).
The PCR fragment obtained was digested with SapI and ApaI and subcloned
into plasmid p83-2 (17), which contains an HIV-1NL4-3backbone. Two different
clones were selected where the only difference was the presence of a threonine
or an asparagine at Gag residue 242.
Site-directed mutagenesis. The T242N mutant virus was constructed in p83-2
by substituting Asn for Thr at Gag position 242 with the GeneTailor site-directed
mutagenesis system (Invitrogen, Barcelona, Spain). The whole plasmid DNA was
PCR amplified in a mutagenesis reaction with the following two overlapping
primers, one of which contained the target mutation: p24/1497-1526, 5?-A GCA
GGA ACT ACT AGT AAC CTT CAG GAA CA-3? (mutagenesis site is un-
derlined); and p24/1497-1513, 5?-T ACT AGT AGT TCC TGC TAT GTC ACT
TCC CC-3?. The presence of the T242N mutation was verified by DNA sequenc-
ing from nucleotide positions 983 to 1656 of Gag from newly generated plasmid
clones. The DNA fragment ranging from the SapI site (position 1107) to the
ApaI site (position 2010) was then subcloned into a new p83-2 plasmid to avoid
carryover of potentially undesirable mutations in the mutagenized plasmid, and
the p24 coding region sequence was verified again.
Generation of viral stocks. Viral stocks were generated in MT4 cells by
electroporation with the mutant proviral plasmid carrying the 5? half of the
genome (p83-2 and derived mutants) and a plasmid carrying the 3? half of the
genome of HIV-1NL4-3(p83-10). The 50% tissue culture infective dose was
determined for each viral stock on MT4 cells, using the Spearman-Karber
Single-cycle infectivity assay. The infectivity of viral stocks was measured by
the single-cycle infectivity assay with Ghost-CXCR4 cells (17). Briefly, a total of
5 ? 104cells/well were infected in triplicate with 50 ng of p24 antigen equivalents
of virus in the presence of 20 ?g of Polybrene/ml by spin inoculation (3 h at
1,500 ? g and 22°C). The proportion of green fluorescent protein (GFP)-positive
cells was measured by fluorescence-activated cell sorting analysis at 24 h postin-
Analysis of relative replicative fitness in virus mixtures. Growth competition
assays were performed with MT4 cells as described elsewhere (17). Infections
were initiated with unequal amounts of two competing virus variants, according
to virus infectivity titrations, to a multiplicity of infection of 0.001. Unequal ratios
were used based on the rationale that an increase in the proportion of the
initially less abundant virus suggests a relatively better replicative capacity than
that of the competing, initially more abundant virus, although random factors
cannot be excluded with either unequal or equal starting ratios. Five-milliliter
cultures were maintained in six-well tissue culture plates at 0.5 ? 106cells/ml.
Every 3 to 4 days, MT4 cells were reinfected with a 1:100 dilution of the
supernatant and cultured at 0.5 ? 106cells/ml.
Supernatants were removed from the cultures every 3 to 4 days for 60 days and
were stored at ?80°C. Viral RNAs were extracted from the supernatants (Qiamp
RNA mini kit; QIAGEN) and PCR amplified in duplicate in two independent
PCRs. The relative proportions of the two competing variants were determined
at each passage based on the ratios of the specific mutations. Ratios were
estimated based on the relative peak heights in electropherograms obtained by
automated sequencing of HIV-1 p24 from culture supernatants. Genotyping was
performed by using a BigDye Terminator cycle sequencing kit (Applied Biosys-
tems) and subsequent electrophoresis in an automated sequencer (ABI PRISM
3100; Applied Biosystems). MT4 cells were also separately infected with mutants
in the absence of wild-type virus to assess possible spontaneous reversion of the
Isolation, amplification, and sequencing of HIV-1 gag. Sequences for the 206
B*57/5801-positive and 239 B*57/5801-negative subjects were determined from
proviral DNAs by population sequencing (24). All sequencing was carried out
using BigDye V3 ready reaction termination mix (Applied Biosystems).
Statistical analysis. The identification of polymorphisms associated with the
TW10 mutations identified at Gag positions 242, 248, 250, and 252 was under-
taken using Fisher’s exact test (see Fig. 3) for each of these four polymorphisms
against all other polymorphisms in the N-terminal domain of p24 (residues 133
to 283), where the variation in B*57/5801-positive subjects was ?10% of the
consensus. These variations were at Gag positions 138, 146, 147, 163, 165, 215,
219, 223, 228, 256, and 260. Polymorphisms at Gag positions 146, 147, 163, and
165 are previously defined B*57/5801-associated CTL escape mutations (24, 25).
Dominance of T242N escape mutation. Initial studies of
individuals expressing HLA-B*57 and -B*5801 described the
early selection of escape mutations within the epitope TSTL
QEQIGW (Gag residues 240 to 249), arising principally at
residues 242 and 248. In an extended analysis to examine the
alternative TW10 mutational escape strategies available to
HIV-1, two large cohorts of B- and C-clade-infected persons
were studied, including 206 B*57/5801-positive subjects (116
B*57-positive and 90 B*5801-positive subjects) (Table 1) and
239 B*57/5801-negative subjects. The T242N mutation arose in
74% (153/206) of B*57/5801-positive subjects. Substitutions at
Gag residue 248, usually G248A, mainly arose in B clade in-
fections (61% [49/80] of subjects) and arose in only a minority
(15%) of C-clade-infected subjects, where Ala-248 was the
T242N mutation reduces viral replicative capacity. The
principal B*57/5801-TW10 escape mutation, T242N, is found
transiently or not at all in B*57/5801-negative subjects, since
transmission of this T242N variant to a B*57/5801-negative
subject was followed by reversion to the wild-type residue T242
(24). In order to determine whether this implied cost of the
T242N mutation to viral replicative capacity is observed in
vitro, the replication of NL4-3 was compared in competition
assays with that of a virus differing only in the T242N substi-
tution (Fig. 1A). In addition, an NL4-3 virus containing the gag
sequence obtained from a B*57-negative child in whom rever-
sion to the wild-type residue T242 subsequently occurred was
compared to a virus differing only in the T242N mutation (Fig.
1B). In each case, the wild-type virus outgrew the T242N vari-
ant. These data support the earlier inference of a fitness cost of
the T242N mutation from observations of reversion in B*57/
5801-negative subjects (24).
The extent of the reduced viral fitness due to the T242N
mutation evident from in vitro competition experiments was
not, however, consistently seen in single-cycle replication as-
says with GFP-expressing cells (Fig. 1C). This suggests that the
T242N mutation does not cripple the virus but acts more subtly
to partially disable it. These data are consistent with the ob-
servation that replacement of the T242N mutant virus by a
T242-containing wild-type virus takes tens of months (24).
Structural role of T242. In order to understand the struc-
tural constraints on the Gag capsid, it is useful to consider the
multiple roles of this polypeptide during the virus life cycle.
The Gag capsid (CA; Gag residues 133 to 363) comprises two
3618MARTINEZ-PICADO ET AL.J. VIROL.
predominantly ?-helical domains, i.e., an N-terminal (CAN;
residues 133 to 283) and a C-terminal (residues 284 to 363)
domain. The B*57/5801-TW10 epitope (Gag residues 240 to
249) thus lies within the N-terminal domain. Following cleav-
age of Gag matrix (MA; Gag residues 1 to 132), the capsid
rearranges into the characteristic cone-shaped core structure
packaging the RNA genome within the virion. The N terminus
of CAN(Fig. 2) forms a beta-hairpin structure subsequent to
Gag cleavage, triggering capsid assembly via the formation of
a salt bridge between the new N-terminal amide group and a
buried carboxyl group (Asp-183). CANis also responsible for
packaging copies of the host protein cyclophilin A (CypA; a
prolyl isomerase and chaperone protein), which is essential for
HIV infectivity (8).
Structures are available for mature CAN(7, 28), immature
CAN(a construct incorporating MA) (31), and a complex of
FIG. 1. Fitness cost resulting from T242N mutation. (A) Competi-
tion experiments in MT4 cells between HIV-1NL4-3and its site-directed
T242N mutant. (B) Competition experiments in MT4 cells between
p24 recombinant viruses from an HLA-B*57-negative child (SMH-05-
Child) who was vertically infected by his HLA-B*57-positive mother.
The only difference between both viral recombinants is the presence of
Thr or Asn at Gag residue 242. (C) Infectivities of individual viral
isolates, as assessed with Ghost reporter cells. The rate of infectivity is
expressed as the percentage of GFP-positive cells at 24 h postinfection.
The results shown represent the means ? standard deviations from
TABLE 1. Patterns of sequence variation in HLA-B*57-
and -B*5801-positive subjects within Gag epitope TW10
(residues 240 to 249) in B and C clade infections
Infection type and
No. of subjects expressing allele
B*57 B*5801 B*57/5801
132 4878 126
VOL. 80, 2006 HIV-1 FITNESS COST OF CTL ESCAPE3619
CANwith CypA (14) (Fig. 2). Comparison of these structures
shows that the N-terminal beta-hairpin formation is coupled to
structural changes at the distal CypA binding site via a 2-Å
displacement of helix 6 (Fig. 2a) (31). This conformational
coupling is relevant to the T242N mutation, since Thr-242 caps
the N terminus of helix 6, with the side chain hydroxyl hydro-
gen bonding to the amide nitrogen of Glu-245 in the first turn
of the helix and the side chain carboxylate of Glu-245 forming
a reciprocal hydrogen bond to the amide nitrogen of residue
242, thereby forming a classic TXXE N-capping box (Fig. 2b)
(3). Residue 242 is thus critical to nucleating helix 6 and sta-
bilizing the electrostatic charge along the helix. Mutation of
this residue to Asn will not abolish its ability to cap the helix,
but the lengthened side chain will reduce its stabilizing
effect, consistent with a viable phenotype of somewhat re-
This pattern of escape and reversion observed for the T242N
mutation is also seen at position 2 in the CTPYDINQM (CM9)
epitope of SIV Gag (SIV Gag residues 181 to 189, with T182
corresponding to T180 in HIV-1 Gag ). When the SIV
epitope is modeled on the HIV-1 structure, Thr-180 also forms
an N-capping box, in this case TXXD, with Asp-183 (as de-
scribed above; involved in triggering assembly). Thr-180 is in
close proximity to Thr-242 (Fig. 2c), and together they span the
bases of the N-terminal hairpin and the CypA binding loop.
Compensatory mutations, most commonly I159V and I204V,
but also some within the TW10 epitope, are required to ac-
commodate the T180A escape mutation (13). It appears that
the critical role of the side chains involved in N-capping of the
helices, which substitute for main-chain hydrogen bonds to
precisely define the start point of the secondary structure,
ensures that escape mutations within epitopes associated with
control of viremia incur a fitness cost.
To consider the impact of the T242N mutation on capsid
protein function, destabilization of helix 6 may disturb the
conformational coupling between the N-terminal hairpin, helix
6, and the CypA binding loop. Additionally, the carboxylate of
Glu-245 forms a second hydrogen bond directly with Arg-229,
which is part of the CypA binding loop (Fig. 2b). Residue 242
is therefore central to a delicate series of interactions, changes
in which might perturb CypA binding. This is supported by the
location of the T242N compensatory mutation, H219Q, also
within the CypA binding loop.
FIG. 2. Structural basis for fitness cost of T242N mutation. (a)
Superimposition of capsid N-terminal domain structures. Gray rope,
immature capsid structure (6, 31); purple rope, mature capsid struc-
ture (6, 31); green ribbon, CypA-bound structure (6, 14); cyan ribbon,
CypA. Gray arrows indicate the observed conformational movements
in the N-terminal hairpin, the CypA binding loop, and helix 6. Residue
T242 is shown in magenta and drawn in stick representation. (b)
Close-up of the TW10 epitope (magenta). The T(242)XXE(245) N-
terminal cap for helix 6 is shown, with hydrogen bonds drawn as yellow
dashes. Side chains (6) are drawn in stick representation. Hydrogen
bonds between Trp-249, Glu-245, and the base of the CypA loop are
shown together with Gly-248. (c) Ribbon representation as in panels a
and b, with the mature capsid structure (6) shown as an orange rope
with side chains depicted as sticks. The residues conferring escape are
shown (T242, G248, N252, and M250). Note the close proximity of
T242 to T180 (corresponding to T182 in SIV). T180 also forms an
N-capping box; in this case, TXXD(183) stabilizes helix 3 and D183
also stabilizes the N-terminal hairpin. Residues I256 and M228, at
which compensatory mutations occur, are also shown as sticks.
3620 MARTINEZ-PICADO ET AL.J. VIROL.
“Compensatory” and alternative escape mutations associ-
ated with TW10 escape. Escape within TW10 via T242N mu-
tation and within CM9 via T180A mutation, with both epitopes
associated with the control of viremia, is associated with addi-
tional variants that are hypothesized to compensate for the
cost of the escape mutation (13, 24). Such “compensatory”
mutations were first described in relation to a third p24 Gag
epitope associated with successful control of HIV-1, the HLA-
B27-KK10 HIV-1-specific epitope (20). We hypothesized that
since the dominant TW10 escape mutation, T242N, exacts an
in vitro fitness cost, alternative TW10 escape mutations would
likewise reduce fitness and therefore require compensatory
changes to be selected.
Additional TW10 escape mutations, in C clade infections
only, were identified from the observation that a significantly
larger number of B*5801-positive subjects have no escape mu-
tation within TW10 than B*57-positive subjects (18/78 versus
1/48; P ? 0.001). A flanking mutation downstream of the C
terminus, either M250I or S252N, is associated with a lack of
variation within TW10 (P ? 1.0 ? 10?5and P ? 0.044, respec-
tively) (Fig. 3). The mechanism driving the acquisition of the
M250I and S252N mutations is unknown and is the subject of
a separate study. However, the association of these mutations
with a lack of escape within TW10 suggests that these may
represent processing escape mutations, as recently described
for HIV infection (1, 9, 33).
To examine the possibility that the TW10 mutations at Gag
residues 248, 250, and 252 might also select for compensatory
mutations, associations between these polymorphisms and
others within the capsid protein were sought (Fig. 3). For each
of these, a strong association with a potential compensatory
mutation(s) was identified (Fig. 3). For example, for B*57/
5801-positive subjects, variation at Gag residue 248 in C clade
infections was observed in association with variation at Val-256
in 20/20 cases, whereas wild-type Ala-248 was associated with
variation at Val-256 in 65/106 subjects (P ? 0.0002). Moreover,
these associations persisted in B*57/5801-negative subjects,
both suggesting a lack of reversion following transmission and
providing strong evidence for the interdependence of appar-
ently minor sequence changes within this protein. For example,
Ala-248 variation is also associated with Val-256 variation in
B*57/5801-negative subjects (P ? 0.0011); for all subjects,
therefore, irrespective of HLA type, the association between
variation at Ala-248 and that at Val-256 is strong (P ? 3.1 ?
10?7). These data suggest that evasion of the TW10 CTL
response is severely limited by restrictions on sequence
changes allowable within p24 Gag.
The one exception to this rule is the G248A mutation within
TW10, which occurs at little or no cost to the virus: the G248A
mutation arises commonly in B clade infections, persists upon
transmission to B*57/5801-negative subjects (24), is not asso-
ciated with a putative compensatory mutation, and actually
increases infectivity (32). However, on its own, it is not an
effective escape variant. The G248A change alone brings about
a partial loss of recognition of the epitope, while the T242N
mutation alone causes a greater loss of recognition, and the
combination of the T242N and G248A mutations completely
abrogates recognition at low peptide concentrations (24). In C
clade infections, however, where Ala-248 is the consensus, the
escape A248X mutation is relatively uncommon, arising only in
association with the V256I mutation, as described above. An
A248G mutation as an escape mutation in C clade infections is
notably rare (Table 1), consistent with the evidence that the
G248A mutation increases rather than decreases infectivity
(32). These data suggest that this commonly arising G248A
change in B clade infections arises at little or no cost to viral
replicative capacity but is relatively ineffective as an escape
variant except in company with the T242N mutation.
Structural analysis indicates that Gag residue 248 is on the
side of helix 6 in proximity to the CypA binding loop. Indeed,
when CypA is bound, Met-228 in the CypA binding loop packs
against Gly-248. Thus, variants at Gag residue 248 are likely to
have an effect on the packing of the CypA binding loop, al-
though since Gag residue 248 is Ala in wild-type clade C, this
FIG. 3. Polymorphisms within the Gag p24 N-terminal domain associated with TW10-linked mutations at Gag residues 242, 248, 250, and 252.
* For H219X, X ? Q in 71 cases, P in 2, and R in 1; for A248X, X ? 20T, 5G, 3Q, 3N, 4D, and 1I. † Data for 2 by 2 tables is represented as
[47/106/1/52], where 47 subjects had T242N and H219X, 106 subjects had T242N and H219, 1 subject had T242 and H219X, and 52 subjects had
T242 and H219.
VOL. 80, 2006 HIV-1 FITNESS COST OF CTL ESCAPE3621
effect is apparently not deleterious. The importance of these
finely balanced interactions is further emphasized by the ob-
servations that the V256I mutation is tightly linked with A248
variants in C clade infections and that the M228L mutation is
similarly strongly associated with the S252N mutation (Fig. 2c
This study addresses one of the central conflicts in the en-
gagement between HIV and the immune response, namely, the
balance between the opposing forces driving mutational escape
and those driving sequence conservation: this may be decisive
in influencing outcomes from HIV-1 infection. HLA-B*57 and
HLA-B*5801 are associated with effective control of HIV-1
(21), and a proposed mechanism to contribute to this instance
of successful immune control (24) is that B*57/5801-restricted
CTL target a conserved epitope, TW10, from which escape
comes only at a cost to viral replicative capacity. This study
demonstrates an in vitro fitness cost resulting from the T242N
mutation. Alternative escape options for the virus are limited,
given that all alternative sites of TW10 escape are associated
with linked mutations, suggesting required functional compen-
sation. The mechanisms for these associations between TW10
escape mutations and their linked polymorphisms are pres-
ently unknown. However, these structural data indicate the
critical role of Thr-242 in nucleating and stabilizing helix 6 in
the capsid protein and highlight the interdependence of key
residues within TW10, helix 6, and the CypA loop to explain
the observed inflexibility of sequence changes in this region. As
with other epitopes associated with successful HIV/SIV control
(4, 13, 14, 19), the capsid protein is a promising target for
effective CTL, perhaps because of selection pressure against
single escape mutations arising in the absence of linked com-
Recent studies from the SIV macaque model of HIV infec-
tion have demonstrated the similar phenomenon of pyrrhic
escape, that is, viral escape from a particular CTL specificity
followed by immune control (26). However, it was noted from
follow-up studies that escape mutation within a single capsid
protein epitope, albeit occurring at a fitness cost, may not be
sufficient to explain the subsequent immune control of immu-
nodeficiency virus enjoyed by those macaques (22). In other
words, HLA-B*57 and -B*5801-restricted CTL responses in
addition to that to TW10 may be required to explain the
successful control of HIV observed in persons expressing these
The putative compensatory mutations that are described in
this study also warrant further discussion. The associations
between particular Gag capsid protein polymorphisms and the
TW10-related escape mutations identified suggest a biological
interdependence between the linked mutations. However, it is
not known how this interdependence operates. Although the
data presented have emphasized a potential structural role for
these reciprocal changes, the compensatory mutations may
operate via a wholly different mechanism. For example, the
H219X mutation observed almost exclusively in association
with the T242N mutation in B*57/5801-positive subjects (Fig.
3) (only 1 of 48 B*57/5801-positive subjects having the H219X
mutation did not have the T242N mutation; P ? 2.5 ? 10?6)
may be selected as a means by which viral replicative capacity
is restored to its original level, but in a way that is independent
of the structural impact of the T242N substitution. Our own
preliminary data (not shown) indicate that the introduction of
the substitution H219Q increases the replicative capacity of the
virus carrying the T242N mutation in vitro. This would be
consistent with a recent paper demonstrating that H219Q and
H219P mutations increase viral replicative capacity indepen-
dent of any change at T242 (15). Similarly, under pressure
from particular protease inhibitors, a necessary requirement
for the replication competence of drug-resistant virus was the
development of substitutions in Gag such as H219Q (16).
Furthermore, these data emphasize the predictable nature
of escape mutations, prompting the hypothesis that prior vac-
cination with both wild-type virus and the anticipated escape
mutants would limit viral escape options even further than in
natural infection. Primary responses in individual subjects can
be generated both to wild-type TW10 and to the escape forms
(10). The absence of TW10 escape is associated with long-term
control of HIV (27), so simultaneously shutting down all viable
escape options may prove a highly successful strategy against
HIV-1, if it can be implemented. The use of structural data to
aid in identification of the limits of the capacity of the virus to
evade potent early CTL responses thus forms a valuable con-
tribution to vaccine design.
This work was funded by the NIH (contracts NO1-A1-15422 and
A146995-01A1) and the Wellcome Trust (A.L. and P.G.). J.M.-P. has
a contract (99/3132) and J.G.P. has a BEFI fellowship (01/9067) from
the Spanish Ministry of Health. B.W. is a Doris Duke Distinguished
Clinical Science Professor, D.I.S. is an MRC research professor, and
P.G. is an Elizabeth Glaser Pediatric AIDS Foundation scientist.
We thank Robert Esnouf and Paul Klenerman for discussions.
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