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Journal of Virology, December 2003, p. 12572-12578, Vol. 77, No. 23
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.23.12572-12578.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Simian-Human Immunodeficiency Virus Escape from Cytotoxic T-Lymphocyte Recognition at a Structurally Constrained Epitope

Fred W. Peyerl,¹ Dan H. Barouch,¹ Wendy W. Yeh,¹ Heidi S. Bazick,¹ Jennifer Kunstman,² Kevin J. Kunstman,² Steven M. Wolinsky,² and Norman L. Letvin^1*

Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215,¹ Department of Medicine, Medical School, Northwestern University, Chicago, Illinois 60611²

Received 14 July 2003/ Accepted 29 August 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
Virus-specific cytotoxic T lymphocytes (CTL) exert intense selection pressure on replicating simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) in infected individuals. The immunodominant Mamu-A^*01-restricted Gag p11C, C-M epitope is highly conserved among all sequenced isolates of SIV and therefore likely is structurally constrained. The strategies used by virus isolates to mutate away from an immunodominant epitope-specific CTL response are not well defined. Here we demonstrate that the emergence of a position 2 p11C, C-M epitope substitution (T47I) in a simian-human immunodeficiency virus (SHIV) strain 89.6P-infected Mamu-A^*01⁺ monkey is temporally correlated with the emergence of a flanking isoleucine-to-valine substitution at position 71 (I71V) of the capsid protein. An analysis of the SIV and HIV-2 sequences from the Los Alamos HIV Sequence Database revealed a significant association between any position 2 p11C, C-M epitope mutation and the I71V mutation. The T47I mutation alone is associated with significant decreases in viral protein expression, infectivity, and replication, and these deficiencies are restored to wild-type levels with the introduction of the flanking I71V mutation. Together, these data suggest that a compensatory mutation is selected for in SHIV strain 89.6P to facilitate the escape of that virus from CTL recognition of the dominant p11C, C-M epitope.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Since cytotoxic T lymphocytes (CTL) play a central role in containing simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) replication in infected individuals (17, 20, 30), dominant epitope-specific CTL exert considerable selection pressure on replicating virus (1-3, 5, 7, 9, 11, 15, 18, 20, 25-27, 36). This selection pressure is so great that a dominant Tat epitope universally mutated away from CTL recognition during the period of primary SIVmac239 infection in Mamu-A^*01⁺ rhesus monkeys (1). Moreover, mutations in dominant Gag and Env epitopes occurred in seven of nine Mamu-A^*01⁺ rhesus monkeys following SIVsmE660 challenge during the assessment of a DNA vaccine strategy (2). In fact, regions of HIV type 1 (HIV-1) hypervariability have been shown to be associated with specific CTL epitopes in humans with particular major histocompatibility complex class I haplotypes (24). Thus, at the level of an individual as well as a population, CTL shape the evolution of SIV and HIV variations.

However, some regions of SIV and HIV proteins are highly conserved among virus isolates, presumably due to constraints dictated by structural requirements for viral replication competence. The gp41 protein is 92% conserved among all sequenced isolates of HIV-1. Moreover, a 20-amino-acid sequence in the capsid of the HIV-1 Gag protein is highly conserved among all known retroviruses (35). Since the determinants of epitope selection are not associated with particular structural requirements of the virus, structural constraints should limit some dominant epitopes from mutation away from CTL recognition. The strategies used by SIV and HIV to balance conflicting selection pressures for viral escape from CTL on the one hand and viral structural competence on the other hand remain poorly defined.

Monkey 798 was a member of a cohort of animals vaccinated with cytokine-augmented DNA immunogens and subsequently infected with simian-human immunodeficiency virus (SHIV) strain 89.6P (SHIV-89.6P) (4). Viral sequence analysis of the immunodominant Gag CTL epitope p11C, C-M in this monkey revealed the emergence of a position 2 mutation at week 20 following infection (3). This single nucleotide mutation, resulting in a threonine-to-isoleucine substitution (T47I), led to significantly less binding of the mutant p11C, C-M epitope peptide (CIPYDINQM) to the Mamu-A^*01 molecule than of the wild-type p11C, C-M epitope peptide (CTPYDINQM), allowed the virus to escape from recognition by p11C, C-M-specific CTL, and led to clinical disease progression in this monkey. Why this was the only monkey in the experimental cohort of SHIV-89.6P-infected Mamu-A^*01⁺ monkeys to demonstrate this phenomenon of escape at the p11C, C-M epitope was unclear.

The p11C, C-M epitope lies within a region of Gag that is highly conserved among characterized HIV-1, HIV-2, and SIV isolates. In fact, recent studies have demonstrated that specific residues of this epitope are required for optimal viral replication (29, 33). Therefore, structural constraints may limit SHIV-89.6P from mutating away from p11C, C-M-specific CTL recognition. In the present study, we evaluated the functional consequences of the T47I mutation for SIV replication and explored the possibility that compensatory viral mutations distant from this immunodominant epitope are required to facilitate viral escape from the p11C, C-M-specific CTL response.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Virus sequencing. Virus sequence analyses of 500-bp regions of gag were performed as described previously (36). Virus from frozen plasma samples was isolated by centrifugation at 25,000 x g for 1 h and lysed with 48% guanidine thiocyanate-1.4% dithiothreitol-1% N-lauroylsarcosine-1% sodium citrate. RNA was precipitated with isopropanol and solubilized. The positions of the oligonucleotide primers are numbered according to the complete SIV isolate 239 proviral genome and flanking sequence (GenBank accession no. M33262) (19). First-strand cDNA was synthesized with reverse transcriptase (RT) by using an SIV gag primer with the sequence 5'-TGTTTGTTCTGCTCTTAAGCTTTTGTAG-3' (nucleotides [nt] 2211 to 2238). Initial PCR amplification was performed with the following primers: gag forward, 5'-ACCTAGTGGTGGAAACAGGAACAG-3' (nt 1628 to 1651), and gag reverse, 5'-TGTTTGTTCTGCTCTTAAGCTTTTGTAG-3' (nt 2211 to 2238). Secondary nested PCR amplification was performed with the following primers: gag forward, 5'-AGCACCATCTAGTGGCAGAGGA-3' (nt 1683 to 1704), and gag reverse, 5'-GAAATGGCTCTTTTGGCCCTT-3' (nt 2171 to 2191). The amplified fragments were cloned into pCRII-TOPO (Invitrogen, Carlsbad, Calif.) or pAMPI (Strategene, La Jolla, Calif.), and individual transformed colonies were subjected to T7-SP6 dideoxy sequencing.

gag point mutations. gag mutants T47I, I71V, and T47I/I71V were created by PCR mutagenesis with a QuikChange kit (Stratagene) and p239SpSp5' as the template; p239SpSp5' was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), from Ronald Desrosiers (19, 28). Oligonucleotide primers T47IF (5'-CAGGCACTGTCAGAAGGTTGCATCCCCTATGACATTAATCAGATGTTAAATTG-3'; nt 1831 to 1883) and T47IR (5'-CAATTTAACATCTGATTAATGTCATAGGGGATGCAACCTTCTGACAGTGCCTG-3'; nt 1831 to 1883) were used to change Thr (ACC) to Ile (ATC). Oligonucleotide primers I71VF (5'-GCGGCTATGCAGATTATCAGAGATGTTATAAACGAGGAGGCTGCAGATTG-3'; nt 1900 to 1949) and I71VR (5'-CAATCTGCAGCCTCCTCGTTTATAACATCTCTGATAATCTGCATAGCCGC-3'; nt 1900 to 1949) were used to change Ile (ATT) to Val (GTT). Double mutant T47I/I71V was created by using the T47I plasmid as a template and oligonucleotide primers I71VF and I71VR.

Plasmids. gag expression plasmids were constructed by PCR amplification of the entire gag gene (nt 1309 to 2841) with primers gagF (5'-AAGAGATCTGCCACCATGGGCGTGAGAAACTCCGTCTTGTCAGG-3'; nt 1309 to 1337) and gagR (5'-GAGAGATCTCTACTGGTCTCCTCCAAAGAGAGAATTGAGG-3'; nt 2811 to 2841) from the wild-type and mutant p239SpSp5' plasmids. To obtain high-level protein expression, a Kozak sequence (GCCACC) was engineered into primer gagF. The amplified gag gene was cloned into expression vector pVRC, provided by G. Nabel (Vaccine Research Center, NIAID, NIH), by using BglII restriction sites that were incorporated into both primer gagF and primer gagR.

Viruses and cells. Single-round reporter viruses (SHIV-CAT) were produced in 293T cells (American Type Culture Collection) by using plasmid pSHIVenvCAT as previously described (32). Viruses were pseudotyped with either the SHIV KB9 (SHIV-KB9) envelope glycoprotein or a mutant envelope glycoprotein (K/S) by using previously described plasmids (6). Plasmid pSHIVenvCAT and the envelope plasmids were kindly provided by J. Sodroski (Dana-Farber Cancer Institute, Boston, Mass.). Recombinant SIVmac239 viruses were produced as previously described (19, 28). Briefly, 5 µg of each proviral half was digested with SphI, phenol-chloroform extracted, ethanol precipitated, and ligated. The ligation mixture was then transfected into CEMx174 cells (American Type Culture Collection) by the DEAE-dextran method. Cultures were monitored for p27 expression, and virus was harvested 7 to 10 days after transfection.

Protein quantification and Western blotting. 293T cells were transfected with 1 µg of purified gag expression plasmid by using a CellPhect transfection kit (Amersham Pharmacia, Piscataway, N.J.). Forty-eight hours later, cell supernatants were collected, and cells were lysed for 20 min with 50 mM Tris-HCl (pH 7.5)-150 mM NaCl-1% Nonidet P-40-0.5% sodium deoxycholate-protease inhibitors (Roche, Mannheim, Germany). Cell lysates were centrifuged for 20 min at 16,000 x g, and the soluble fraction was separated from the insoluble pellet. The insoluble pellet was washed three times with phosphate-buffered saline. To normalize samples, total protein content was quantified by using a Bio-Rad (Hercules, Calif.) protein assay. An SIV core antigen assay (Coulter, Miami, Fla.) was used to quantify the concentrations of SIV p27 in the supernatant and soluble cell lysate. The soluble cell lysate and insoluble cell pellet were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes for Western blotting. An anti-SIVmac p27 monoclonal antibody (55-2F12) was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, from Niels Pederson (16). Primary detection antibody 2F12 was used at a 1:2,000 dilution and detected by using a SuperSignal West Pico mouse immunoglobulin G detection kit (Pierce, Rockford, Ill.).

Single-round infection assay. Rhesus monkey peripheral blood mononuclear cells (PBMCs) were purified by Ficoll-Paque density gradient centrifugation. Contaminating erythrocytes were lysed with a solution containing 150 mM NH₄Cl and 7 mM NaHCO₃ (pH 7.2). Cells were washed three times with phosphate-buffered saline and resuspended in RPMI 1640 complete medium plus 10% fetal calf serum, penicillin, and streptomycin. Cells were then stimulated for 2 days with 6.25 µg of concanavalin A per ml and 20 U of interleukin-2 per ml 24 h prior to infection. Equivalent amounts of virus, measured in RT units, were used to infect 2 x 10⁶ rhesus monkey PBMCs and macrophages overnight. Seventy-two hours following infection, cells were assayed for chloramphenicol acetyltransferase (CAT) expression by using a CAT enzyme-linked immunoassay kit (Roche).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Prospective analysis of SHIV-89.6P gag sequence evolution in vaccinated, infected monkey 798. The p11C, C-M-specific CTL response is immunodominant in SHIV-89.6P-infected, Mamu-A^*01⁺ rhesus monkeys and therefore should exert considerable selection pressure on replicating virus in these animals. However, only one of eight Mamu-A^*01⁺ monkeys vaccinated with DNA-interleukin-2-immunoglobulin developed a p11C, C-M escape mutation during 3 years of monitoring following SHIV-89.6P challenge. Interestingly, analysis of sequence evolution in a 500-bp region of gag in the plasma of that animal (monkey 798) demonstrated the coincident emergence of a p11C, C-M T47I mutation and an isoleucine-to-valine mutation at position 71 (I71V) of the p27 capsid protein (Fig. 1). The coincident T47I and I71V mutations were observed in all 53 viral clones obtained from plasma sampled at weeks 20, 24, 28, 36, and 44 following challenge. These mutations were found in none of the eight viral clones obtained from plasma sampled at week 14 following challenge. In addition, the I71V mutation was found in none of the 16 viral clones isolated from the SHIV-89.6P challenge stock that was used to infect monkey 798 and in none of the viruses sequenced at the set point from the other seven animals in the study. The coincident emergence of the T47I and I71V mutations suggested that there was an important linkage between these sequence changes.

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FIG. 1. Emergence of both a CTL epitope and an associated flanking mutation in the gag gene of SHIV-89.6P from monkey 798. Sequence analysis of the gag gene was performed on 8 to 16 viral clones per time point with sequence-specific primers. The deduced amino acid sequence for a 45-amino-acid segment is shown. The Mamu-A*01-restricted Gag epitope p11C, C-M is shown in bold type and underlined. Numbers in parentheses indicate the number of mutant clones out of the total number of clones analyzed for each time point.

Los Alamos HIV Sequence Database analysis. To analyze further the association between the T47I and I71V mutations, we evaluated 74 SIV and HIV-2 sequences published in the Los Alamos HIV Sequence Database. Of the 74 predicted amino acid sequences, 21 contained a p11C, C-M epitope mutation at position 2 (T47X) and 13 contained the I71V mutation. Importantly, of the 21 sequences containing the T47X mutation, only 8 did not include the I71V mutation. In addition, the I71V mutation was never found to occur in the absence of the T47X mutation. Statistical analysis (Fisher's exact test) revealed a significant association between T47X and I71V (P < 0.0001). Therefore, as seen with the virus from monkey 798, viral sequence analysis with the Los Alamos HIV Sequence Database demonstrated that mutations at position 2 of the p11C, C-M epitope (T47X) are highly associated with a single flanking isoleucine-to-valine mutation at a distance of more than 20 amino acids from this epitope residue.

Contribution of the I71V mutation to viral protein expression. Because of coincident appearance of the T47X and I71V mutations in the predominant virus of monkey 798 and the statistical association between these mutations in sequenced viruses in the Los Alamos HIV Sequence Database, we hypothesized that a selective advantage may exist for viruses with both of these mutations. To explore the functional consequences of these mutations, we generated a plasmid expressing full-length wild-type SIVmac239 gag. In addition, we generated plasmid gag constructs expressing either the p11C, C-M mutation alone (T47I), the downstream valine mutation alone (I71V), or a combination of both mutations (T47I and I71V). We transfected 293T cells with these gag expression plasmids and 48 h later assessed Gag p27 expression in supernatants and soluble cell lysates. Cells transfected with the T47I plasmid had significantly lower p27 expression in both supernatants (P < 0.005) and soluble cell lysates (P < 0.001) than did cells transfected with the wild-type plasmid (Fig. 2A and B). However, protein expression was restored to wild-type levels in both supernatants and soluble cell lysates when this mutation was combined with the I71V mutation (Fig. 2A and B; T47I I71V). The I71V mutation alone appeared to result in a subtle increase in protein expression by the gag plasmid, as detected in cell lysates (Fig. 2B). Western blot analysis of soluble cell lysates (Fig. 2C) and insoluble cell pellets (Fig. 2D) also showed a marked reduction in Gag protein expression in cells transfected with the T47I plasmid, and this expression was restored by the complementing I71V mutation. The I71V mutation alone appeared to increase Gag expression by the plasmid, as detected by Western blot analysis (Fig. 2D). Thus, a single nucleotide mutation (T47I mutation) significantly reduced Gag protein expression, and the addition of the I71V mutation restored wild-type levels of protein expression.

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FIG. 2. Mutation I71V in Gag restores the level of protein production to that of wild-type Gag. 293T cells were transfected with 1 µg of each noted plasmid; 48 h later, the cells were lysed and pelleted. (A and B) SIV Gag p27 levels in supernatants (A) and soluble cell lysates (B) were determined by an ELISA. (C and D) Western blotting was performed to assess soluble cell lysates (C) and insoluble cell pellets (D). Full-length unprocessed Gag is 55 kDa. Each sample was normalized for total protein content by using the Bio-Rad protein assay. The plasmids assessed were as follows: VRC (or VRC 2000), parental plasmid backbone; WT, wild-type SIVmac239 gag; T47I, p11C, C-M threonine-to-isoleucine mutant gag; I71V, isoleucine-to-valine mutant gag; T47I I71V (or T47I, I71V), double-mutant gag. Values illustrated are mean of triplicates ± standard error. All values were compared to those for the wild type. A single asterisk indicates a P value of <0.001 (ANOVA with Bonferroni multiple comparisons).

Contribution of the I71V mutation to viral infectivity and replication. Because the introduction of the above-described mutations significantly altered Gag protein expression by the plasmids, we explored the effect of these mutations on viral infectivity and replication kinetics. We introduced the T47I and I71V mutations alone and in combination into single-round SHIV particles that contained the Escherichia coli CAT gene in place of the envelope gene. 293T cells were transfected with each of the resulting SHIV-CAT plasmids, and the virus produced was pseudotyped with either the HIV-1 KB9 envelope glycoprotein or a mutant HIV-1 envelope glycoprotein that does not support virus entry (K/S). Seventy-two hours later, virus was harvested, and the SHIV-CAT constructs were used to infect rhesus monkey PBMCs. The PBMCs were assayed for CAT expression by an enzyme-linked immunosorbent assay (ELISA) 72 h following infection. As expected, cells infected by viruses pseudotyped with the K/S mutant envelope glycoprotein did not have detectable CAT expression, while cells infected with wild-type SHIV-CAT pseudotyped with the KB9 envelope glycoprotein had readily detectable CAT expression (Fig. 3). Interestingly, cells infected with T47I mutant SHIV-KB9 had nearly background levels of CAT expression, while cells infected with SHIV-KB9 containing both the T47I and the I71V mutations had levels of CAT expression comparable to those of cells infected with the wild-type SHIV-KB9 construct. The I71V mutation in isolation did not alter viral infectivity, since cells infected with I71V mutant SHIV-KB9 had levels of CAT expression comparable to those of cells infected with the wild-type SHIV-KB9 construct. Therefore, the T47I mutation resulted in a dramatic decrease in viral infectivity that was compensated for by the additional I71V mutation.

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FIG. 3. Mutation I71V in Gag T47I restores the infectivity of the CTL escape virus. Rhesus monkey PBMCs were stimulated for 48 h with concanavalin A and then infected overnight with recombinant SHIV-CAT pseudotyped with the K/S or KB9 envelope glycoprotein and containing the noted Gag mutations. CAT activity was measured in cell lysates by an ELISA 72 h following infection. The plasmids assessed were as follows: WT, wild type; T47I, p11C, C-M threonine-to-isoleucine mutant gag; I71V, isoleucine-to-valine mutant gag; T47I, I71V, double-mutant gag. Values illustrated are mean of three experiments ± standard error. All values were compared to those for the wild type. An asterisk indicates a P value of <0.05 (ANOVA with Bonferroni multiple comparisons).

Because subtle differences in virus production and infectivity can have dramatic effects on the fitness of a virus, we analyzed the replication kinetics of SIVmac239 clones incorporating the gag mutations. CEMx174 cells were infected with these SIVmac239 mutants, and supernatants were analyzed for Gag p27 and RT activity (Fig. 4). Cells infected with wild-type SIVmac239 exhibited a peak in Gag p27 production and RT expression on day 6 of culturing, while cells infected with either the T47I or the I71V mutant virus exhibited a delay in the peak expression of Gag p27 and RT, although by day 10 their levels of Gag p27 and RT expression were indistinguishable from those of the wild type. Interestingly, cells infected with the cloned virus incorporating both the T47I and the I71V mutations demonstrated kinetics of Gag p27 and RT expression indistinguishable from those of cells infected with wild-type SIVmac239. Because viral mutations can arise quickly in vitro, we sequenced a 500-bp region of the gag gene at day 10 and found no additional accumulation of mutations or any reversion back to the wild type. Thus, the single mutations T47I and I71V were associated with a decreased level of viral fitness, while the virus incorporating both mutations exhibited replication kinetics comparable to those of the wild-type virus.

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FIG. 4. Mutation I71V restores the rate of replication of the CTL escape virus to that of the wild-type virus. CEMx174 cells were infected with equal amounts of various SIVmac239 clones (wild type; T47I; I71V; and T47I, I71V), and supernatants were monitored for SIV p27 antigen (A) and RT activity (B). Values illustrated are mean of triplicates ± standard error.

Evolution of the virus with T47I and I71V mutations in monkey 798. The virus incorporating both the T47I and the I71V mutations could have evolved by developing the mutations simultaneously, sequentially, or through a recombination event. To explore the events leading to the generation of this virus with both mutations, we sequenced virus in plasma obtained from monkey 798 at week 16 postinfection, 4 weeks before the documented emergence of the T47I/I71V virus (Fig. 1). Sequence analysis of the 500-bp region of gag revealed four different virus species. One of the 15 sequenced clones was wild type, 3 of the 15 contained the T47I mutation alone, 2 of the 15 contained the I71V mutation alone, and 9 of the 15 contained both the T47I and the I71V mutations (Fig. 5). Documentation of clones with each of the single mutations (T47I or I71V) suggests that the virus with both mutations did not arise by simultaneously mutating both positions. Rather, the observations support the possibility of either the sequential accumulation of mutations or a recombination event creating the virus with both mutations. That the majority of the cloned virus fragments contained both mutations is consistent with the notion that this virus has a selective replication advantage.

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FIG. 5. Sequence analysis of plasma virus sampled at week 16 from monkey 798. Sequence analysis of 15 viral clones from a week 16 plasma sample, 2 weeks after analysis of the plasma virus quasispecies demonstrated only the wild-type gag sequence. The deduced amino acid sequence for 45 amino acids is shown. The Mamu-A*01-restricted epitope p11C, C-M is shown in bold type and underlined. Numbers in parentheses indicate the number of mutant clones out of the total number of clones analyzed.

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study delineates the structural constraints on SHIV-89.6P escape from CTL specific for the immunodominant Gag p11C, C-M epitope in Mamu-A^*01⁺ rhesus monkeys. An analysis of viral sequence evolution demonstrated the coincident emergence in a monkey of a residue 2 p11C, C-M substitution and a flanking isoleucine-to-valine substitution at position 71 of the capsid protein. We showed that the p11C, C-M mutation alone was associated with profound decreases in Gag protein expression and viral replication. However, these virologic parameters were restored to wild-type levels with the introduction of a flanking I71V mutation. Moreover, we showed that double mutant T47I/I71V is rapidly selected for in vivo following the emergence of either single mutation in a viral quasispecies. In fact, an analysis of the Los Alamos HIV Sequence Database revealed that the single mutations T47X and I71V are uncommon, while their appearance in combination is frequent. Together, these data suggest that the I71V mutation facilitates SIVmac239 and SHIV-89.6P escape from the immunodominant Mamu-A^*01-restricted Gag p11C, C-M-specific CTL response.

While the present study showed that the T47I mutation significantly decreased Gag protein expression by a plasmid, we and others did not observe an associated defect in the level of virus production when using full-length provirus plasmids (data not shown) (29, 33). This discordance in findings might be attributable to differences in the experimental systems used for these studies. Gag protein expression was assessed with a plasmid expressing only Gag under conditions that were optimal for detecting subtle abnormalities in protein expression. In contrast, virus was generated in cells transfected with full-length provirus plasmids, a setting in which a variety of mechanisms of SIV and HIV gene expression were operative. Thus, the decreased Gag protein expression might have been compensated for in vitro by Rev-dependent export of viral mRNAs (12, 22, 23) or specific env sequences that were recently shown to enhance Gag protein expression (31).

While SIV and HIV escape from dominant epitope-specific CTL is often observed, the rapidity and frequency of this escape can be highly variable. In Mamu-A^*01⁺ rhesus monkeys, mutation at the Tat SL8 epitope predictably occurs during primary infection with SIVmac239 (1), but escape at the Env TL9 and Gag p11C, C-M epitopes occurs more slowly (2, 3, 8). Importantly, the rate and frequency of viral escape from CTL can differ for different viral isolates. Barouch et al. recently showed that seven of nine Mamu-A^*01⁺ rhesus monkeys infected with SIVsmE660 demonstrated viral escape at both the Env TL9 and the Gag p11C, C-M epitopes (2). In contrast, only 1 of 15 Mamu-A^*01⁺ rhesus monkeys infected with SHIV-89.6P demonstrated viral escape from p11C, C-M-specific CTL (3).

A number of factors could influence the rate and frequency of viral escape from CTL and contribute to the striking difference between the cohorts of SIVsmE660- and SHIV-89.6P-infected monkeys in these vaccine studies. The SIVsmE660-challenged monkeys received only a plasmid gag DNA vaccine, while the SHIV-89.6P-infected monkeys received cytokine-augmented plasmid gag and env DNA vaccines. Therefore, the potency of the vaccine-elicited CTL responses was lower in the monkeys eventually challenged with SIVsmE660 than in those challenged with SHIV-89.6P (2, 4, 10). In the setting of less potent CTL responses, higher levels of viral replication and an associated generation of larger numbers of new mutant viruses would be expected. It is also possible that differences in the gene sequences of the challenge viruses might affect the likelihood of developing CTL escape mutations. We have shown in the present study that a compensatory mutation downstream from the p11C, C-M epitope facilitates the generation of a replication-competent virus. While SIVsmE660 in the challenge stock used in the vaccine study did not have this I71V gag mutation, the virus may carry other mutations that will facilitate the generation of viable SIV when a p11C, C-M mutation occurs.

Other factors have also been suggested to influence the rate of development of CTL escape mutations. If, as some have suggested, CTL initially target early gene products, such as Tat and Nef, and only later in the course of infection target structural gene products (14), then the selection pressure will be greatest on early gene products during primary infection and on structural proteins during chronic infection. It has been suggested that early high-avidity CTL specific for Tat and Nef select for escape viruses more frequently than do late low-avidity CTL specific for Gag (26). This paradigm has, however, not been confirmed in in vitro studies with CTL clones with different avidities for the same epitope (37).

Unlike the accessory proteins Tat and Nef, which can retain function even with numerous mutations and large deletions, the structural Gag protein has limited plasticity. The Gag p11C, C-M epitope (CTPYDINQM) is embedded within a highly conserved region of the capsid protein (Fig. 6A), beginning amino-terminal to and ending within helix 3 (amino acid residues 46 to 54). In addition, the Thr, Pro, Asp, Asn, and Met residues of the epitope are found in 201 of the 207 published HIV-1, HIV-2, and SIV Gag sequences in the Los Alamos HIV Sequence Database. This region of Gag has been shown by many groups to play a vital role in HIV and SIV replication. First, a structure-function analysis by von Schwedler et al. showed that, following proteolysis, the amino-terminal end of the capsid refolds into a ß-hairpin or helix structure that is stabilized by the formation of a salt bridge between Pro¹ and Asp⁵¹ (33). Furthermore, the crystal structure of the NH₂-terminal domain of HIV-1 p24 revealed the presence of a hydrogen bond between highly conserved Thr⁴⁸ and Asp⁵¹ (Fig. 6B). In addition, the region encompassing helices 1 to 3 was recently shown to be required for mature capsid formation (34). Finally, mutations that disrupt the formation of a salt bridge were shown to create noninfectious viruses with spherical cores.

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FIG. 6. Analysis of the HIV-1 Gag crystal structure. (A) Ribbon diagram of the NH2-terminal domain of HIV-1 p24. The region involved in the formation of the salt bridge is shown in the dashed box. Side chains for residues Pro1, Thr48, and Asp51 are shown in blue, grey, and green, respectively. Flanking valine mutations are located within helix IV (I72; side chain is located on back and is not visible in this view). (B) Close-up view of hydrogen bond between Thr48 and Asp51. (C) Predicted structure of the T48I mutant. The mutation of Thr48 to Ile48 is predicted to eliminate a hydrogen bond. Models were generated by using Swiss PDB Viewer and the reported crystal structure (13).

It is likely that mutations within the Gag p11C, C-M epitope interfere with virus assembly; additional mutations may be required to compensate for this interference. In support of this notion, computer modeling has demonstrated that mutation of Thr⁴⁸ to Ile⁴⁸ would eliminate the hydrogen bond with Asp⁵¹ (Fig. 6C). We hypothesize that this hydrogen bond is required to stabilize the tertiary structure of the monomeric capsid following its proteolytic cleavage from the matrix. It is possible that the destabilized monomeric capsid can be stabilized by intermolecular hydrophobic interactions between capsids in which Ile⁷¹ has been mutated to Val⁷¹. This possibility is supported by crystallographic models, mutational analysis, and chemical cross-linking experiments showing that helices 1 to 3 are involved in stabilizing capsid hexamers (33) and that helix 4 may promote the assembly of hexameric oligomers (21).

Successful SIV and HIV escape from CTL recognition represents a balance between the selection pressures exerted by CTL and a requirement for viral structural competence. While specific mutations were previously shown to be associated with successful HIV-1 escape from an immunodominant Gag HLA-B27-restricted CTL response, this association has been shown only by sequence and phylogenetic analyses (18). Here we have demonstrated that SHIV-89.6P escape from p11C, C-M-specific CTL requires both a position 2 epitope mutation (T47I) and a compensatory mutation located more than 20 amino acids downstream of this epitope mutation (I71V). Moreover, we have shown that the T47I mutation alone results in decreased viral Gag expression, infectivity, and replication kinetics, while the addition of the I71V mutation restores these deficiencies to the levels of the wild-type virus. While it is unclear whether a requirement for compensatory mutations for viral escape from CTL recognition at epitopes within conserved structural proteins is universally required, this finding of both a p11C, C-M epitope mutation and an associated compensatory mutation in SHIV-89.6P demonstrates the remarkable ability of lentiviruses to adapt to their immunologic environment.

 
We thank J. Sodroski for reagents and helpful conversations and G. Nabel for reagents.

This work was supported by NIH grant AI20729.

 
* Corresponding author. Mailing address: Harvard Medical School, Beth Israel Deaconess Medical Center, Division of Viral Pathogenesis, Research East Room 113, 330 Brookline Ave., Boston, MA 02215. Phone: (617) 667-2766. Fax: (617) 667-8210. E-mail: nletvin{at}bidmc.harvard.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, December 2003, p. 12572-12578, Vol. 77, No. 23
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.23.12572-12578.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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