Gag Cytotoxic T Lymphocyte Escape Mutations Can Increase Sensitivity of HIV-1 to Human TRIM5, Linking Intrinsic and Acquired Immunity

Transcription

1 JOURNAL OF VIROLOGY, Nov. 2011, p Vol. 85, No X/11/$12.00 doi: /jvi Copyright 2011, American Society for Microbiology. All Rights Reserved. Gag Cytotoxic T Lymphocyte Escape Mutations Can Increase Sensitivity of HIV-1 to Human TRIM5, Linking Intrinsic and Acquired Immunity Emilie Battivelli, 1,2 Julie Migraine, 1,2 Denise Lecossier, 1,2 Patrick Yeni, 3 François Clavel, 1,2 and Allan J. Hance 1,2,3 * INSERM U941, Paris 75010, France 1 ; Institut Universitaire d Hématologie, Université Paris Diderot, Hôpital Saint-Louis, Paris 75010, France 2 ; and Service des Maladies Infectieuses et Tropicales, Assistance Publique Hôpitaux de Paris, Hôpital Bichat Claude Bernard, Paris 75018, France 3 Received 24 May 2011/Accepted 1 September 2011 Although laboratory-adapted HIV-1 strains are largely resistant to the human restriction factor TRIM5 (htrim5 ), we have recently shown that some viruses carrying capsid (CA) sequences from clinical isolates can be more sensitive to this restriction factor. In this study we evaluated the contribution to this phenotype of CA mutations known to be associated with escape from cytotoxic T lymphocyte (CTL) responses. Recombinant viruses carrying HIV-1 CA sequences from NL4-3 and three different clinical isolates were prepared, along with variants in which mutations associated with CTL resistance were modified by site-directed mutagenesis, and the infectivities of these viruses in target cells expressing htrim5 and cells in which TRIM5 activity had been inhibited by overexpression of TRIM5 were compared. For both htrim5 -sensitive viruses studied, CTL-associated mutations were found to be responsible for this phenotype. Both CTL resistance mutations occurring within HLA-restricted CA epitopes and compensatory mutations occurring outside CTL epitopes influenced htrim5 sensitivity, and mutations associated with CTL resistance selected in prior hosts can contribute to this effect. The impact of CTL resistance mutations on htrim5 sensitivity was context dependent, because mutations shown to be responsible for the TRIM5 -sensitive phenotype in viruses from one patient could have little or no impact on this parameter when introduced into another virus. No fixed relationship between changes in htrim5 sensitivity and infectivity was discernible in our studies. Taken together, these findings suggest that CTL mutations may influence HIV-1 replication by modifying both viral infectivity and sensitivity to TRIM5. Downloaded from * Corresponding author. Mailing address: INSERM U941, Hôpital Saint Louis, 1 avenue Claude Vellefaux, Paris cedex 10, France. Phone: Fax: Supplemental material for this article may be found at Published ahead of print on 14 September Cytotoxic T lymphocytes (CTLs), which target virus-infected cells through recognition of viral peptides presented by class I HLA molecules, play a critical role in controlling HIV-1 replication in the infected host (26, 72). Numerous studies have demonstrated that the HLA alleles expressed by infected patients have an important bearing on viral load at set point, the rate of CD4 T cell decline, and progression to AIDS (12, 26, 34). Certain HLA alleles (e.g., B*57, B*27, B*14, and B*52) are more frequent in patients that spontaneously control HIV-1 replication to low levels, reflecting differences in the nature of the HLA-viral peptide interactions permitted by these protective alleles, including which viral sequences (epitopes) are presented and how the epitope binds to the HLA molecule (38, 54). In turn, HIV-1 attempts to escape from this immune pressure by introducing mutations in or near the targeted epitopes that either disrupt the processing of the epitopes, impair their binding to the restricting HLA molecule, or weaken interactions with the T cell receptor (3, 18, 33, 57). Predictable patterns of escape mutations in epitopes recognized by a given HLA allele have been identified in population studies (9, 50). Importantly, resistance mutations in some epitopes can impair viral replicative capacity, and this is observed more frequently for mutations occurring in CA than for those occurring in other viral proteins (42, 44, 49, 71). Thus, resistance mutations in CA epitopes are often accompanied by compensatory mutations that serve to correct the impairment in viral replication produced by the resistance mutations (8, 9, 33, 61, 62). The observations that the presence of Gag CTL resistance mutations correlates with lower viral loads in infected patients and that such mutations revert following transmission of the virus to HLA-discordant recipients indicate that the restoration of viral replicative capacity by compensatory mutations is often incomplete (3, 7, 16, 23, 40, 69). The reason that CTL escape mutations in CA are more likely to be deleterious than those in other viral proteins is not fully understood. The CA is a highly conserved structure, and it is known that many mutations can disrupt optimal CA stability (6, 19, 20). In addition, unlike other viral proteins, the CA is targeted by the intrinsic restriction factor TRIM5 (43, 52, 66, 70). TRIM5 interacts with the mature capsid lattice after its entry into target cells; this interaction can directly promote rapid disassembly of the capsid structure, thereby preventing the completion of reverse transcription (67). In addition, TRIM5 possesses an E3 ubiquitin ligase activity that is amplified following interaction of TRIM5 with the capsid, on April 22, 2018 by guest 11846

2 VOL. 85, 2011 SENSITIVITY OF HIV-1 TO TRIM FIG. 1. Measurement of sensitivity to htrim5 and viral infectivity. (A) Recombinant proviruses containing a deletion in env, expressing Renilla luciferase in place of nef, and carrying the Gag-PR sequence from NL4-3 or the clinical isolate NRC10 were prepared, and VSV-pseudotyped viral stocks were obtained by transfecting 293T cells with the proviral construct and a plasmid expressing the VSV G protein. U373-X4 cells transduced with a control vector causing them to overexpress LacZ (U373-X4-LacZ) or with a vector causing them to overexpress TRIM5 (U373-X4-TRIM5 ) were infected with three dilutions of virus. Luciferase activity (relative light units [RLU]) was measured 40 h after infection and plotted as a function of amount of virus, and the slopes of the curves (RLU/ng p24) were determined. (B) To measure viral sensitivity to htrim5, the ratio of the slope obtained for U373-X4-TRIM5 cells to that obtained for U373-X4- LacZ cells was determined for each viral variant. The htrim5 sensitivity of NRC10 relative to that of NL4-3 was determined by calculating the ratio of these two values. (C) To assess infectivity, measurements of viral infectivity in the target cells in which htrim5 activity had been inhibited by overexpression of TRIM5 (U373-X4- TRIM5 cells) were used; values were expressed as percentages of results obtained for NL4-3 in the same experiment. thereby stimulating a cascade that both promotes innate immune signaling and contributes directly to viral restriction by TRIM5 (35, 55). Restriction exerted by TRIM5 on retroviral replication varies according to the virus and to the host species, reflecting pressure exerted on the TRIM5 gene over the course of mammalian evolution by retroviral infections (31). HIV-1 is strongly sensitive to restriction by rhesus macaque TRIM5 (66). In contrast, HIV-1 was until recently considered largely resistant to human TRIM5 (htrim5 ); results from several groups have shown that laboratoryadapted HIV-1 strains such as NL4-3 show only very modest (approximately 2-fold) sensitivity to htrim5 expressed at physiological levels (27, 32, 65, 68). We have recently shown, however, that HIV-1 carrying CA sequences derived from some clinical isolates are more sensitive to htrim5 than NL4-3 and that increasing the expression of htrim5 in target cells, including CD4 T cells, by treatment with alpha interferon (IFN- ) further increases the activity of htrim5 against these viruses (5). Thus, it is possible that the introduction of mutations in CA in response to CTL pressure, in addition to directly modifying the functional properties of CA, could alter its sensitivity to recognition by TRIM5, a possibility that has been suggested by other authors (29, 49). To study this question, we have evaluated the role played by mutations associated with CTL resistance in producing the increased TRIM5 sensitivity observed for the viruses identified in our previous study that carried CA sequences derived from clinical isolates. We demonstrate that both CTL resistance mutations and compensatory mutations can result in increased viral sensitivity to htrim5, a phenomenon that involves not only mutations likely to have developed de novo in the current host but also mutations selected in prior hosts. The impact of CTL resistance and compensatory mutations on htrim5 sensitivity, however, is strongly dependent on the overall context of the CA sequence in which they occur. These findings suggest that modifications in htrim5 sensitivity resulting from CTL mutations are likely to influence HIV-1 replicative capacity in vivo but that the impacts of this effect are likely to be different for different patients. MATERIALS AND METHODS Cell culture. U373-X4 cells in which htrim5 activity had been inhibited by stable overexpression of untagged TRIM5 and the corresponding control cell line that overexpresses LacZ were established by transduction with plenti6/v5- D-TOPO-based vectors using previously described techniques (5). Two independently derived pairs of these transduced cell lines were studied in parallel in all studies reported here. The infectivity of N-tropic and B-tropic murine leukemia viruses (N-MLV and B-MLV) in IFN- -pretreated U373-X4-TRIM5 cells and U373-X4-LacZ cells was evaluated as previously described (5) before and after completion of these studies. In both cases, U373-X4-LacZ cells continued to strongly inhibit the infectivity of N-MLV, whereas similar titers of N-MLV and B-MLV were observed in U373-X4-TRIM5 cells (data not shown). Viruses. The production of the vesicular stomatitis virus (VSV)-pseudotyped pnl4-3-based recombinant viruses that contain a deletion in env, that express Renilla luciferase in place of nef, and whose Gag-PR sequences were derived from clinical isolates (NRC1, NRC2, and NRC10; accession numbers JN to JN408077) or from NL4-3 has previously been described (5, 45). To create variants of these viruses, the BssHII-ClaI fragment from the proviral plasmid was subcloned into a modified pbluescript vector in which one of the two BssHII sites in the polylinker had been removed and one or more nucleotide substitutions were introduced by the procedures described in Tables S1 and S2 in the supplemental material. The modified BssHII-ClaI fragment was then reinserted into the original plasmid. All constructions were verified by sequencing the entire Gag-PR region of the proviral plasmid. VSV-pseudotyped viral stocks were produced as previously described and stored at 80 C (5, 45). Measurement of viral sensitivity to TRIM5 and infectivity. The procedures used to measure sensitivity to TRIM5 and viral infectivity are summarized in Fig. 1. HIV-1 infectivity was measured by determining luciferase activity in target cells after infection as previously described (5, 45). Briefly, U373-X4 cells over- Patient TABLE 1. Class I HLA genotypes of patients evaluated in this study Class I HLA types HLA-A HLA-B HLA-C NRC10 A*01, A*11 B*27, B*55 C*03, C*06 NRC2 A*23, A*24 B*40, B*57 ND a NRC1 A2, A30 B14, B44 ND a ND, not determined.

3 11848 BATTIVELLI ET AL. J. VIROL. t tests with nonpooled standard deviations (SD) and adjusted for multiple comparisons by the method of Holm (1). Correlations were evaluated using the Pearson test. RESULTS FIG. 2. Annotated sequence of the amino-terminal domain of HIV-1 capsid. Shown is the amino acid sequence of the NL4-3 CA (amino acids 1 to 151). The seven helical regions, as described in reference 22, are boxed and numbered (numbers in circles). The CTL epitopes mentioned in the text are indicated, with the exception of QW9, which is located in the C-terminal CA domain. Amino acids that are commonly mutated to induce resistance to CTLs recognizing these epitopes (red) or to compensate for impaired replication resulting from CTL resistance mutations (green) and which were evaluated in this study are indicated. The amino acids are numbered both according to the amino acids in CA (numbers in parentheses) and Gag. expressing LacZ or TRIM5 were incubated in the presence of 1,000 U/ml IFN- for 24 h to increase the expression of TRIM5 and infected in triplicate with serial 2-fold dilutions of virus (nominally 20, 10, and 5 g p24/ml) in the presence of 2 g/ml DEAE-dextran, and luciferase activity was measured 40 h after infection (Fig. 1A). The actual p24 contents of the virus preparations used to infect the cells were measured in each experiment, and these values were used to calculate slopes. The mutations introduced in CA were shown not to influence quantification of p24 (data not shown). The slope (RLU versus ng of p24) was calculated for each virus in each cell line. For all viral variants, RLU values were linearly related to the amount of virus used to infect the cells. To assess sensitivity to TRIM5 (Fig. 1B), the results were expressed as a ratio and normalized to that obtained for NL4-3 according to the formula (slope for U373-X4- TRIM5 /slope for U373-X4-LacZ)/(slope for NL4-3 in U373-X4-TRIM5 /slope for NL4-3 in U373-X4-LacZ). To evaluate viral replicative capacity, as measured in the target cells in which htrim5 activity had been inhibited by overexpression of TRIM5 (Fig. 1C), the slope of RLU versus amount of p24 obtained for U373-X4-TRIM5 cells was normalized to that obtained for NL4-3 in the same experiment. Statistical analysis. All results are expressed as means standard errors of the means (SEM) unless otherwise indicated. Groups within a single series of experiments were compared by analysis of variance (ANOVA) using the R statistical package (version ). Posttest comparisons were performed using Evaluation of htrim5 sensitivity. To evaluate the effect of changes in the HIV-1 CA on the sensitivity to htrim5, we used VSV-pseudotyped NL4-3-based recombinant viruses that contained a deletion in env, that expressed Renilla luciferase in the place of nef, and that had Gag-protease (Gag-PR) sequences derived from NL4-3 or from clinical isolates NRC1, NRC2, and NRC10. As described below, the CA sequences of these viruses were modified by site-directed mutagenesis, and these viruses were used to infect U373-X4 cells that had been transduced with vectors resulting in the stable expression of either TRIM5 or, as a control, -galactosidase. The truncated TRIM5 isoform forms heterodimers with TRIM5, blocking its activity, and the cells overexpressing TRIM5 did not restrict TRIM5 -sensitive virus, as demonstrated by the similar infectivities of N-MLV and B-MLV viruses in this cell line (5). In the experiments presented here, sensitivity to TRIM5 was expressed as the ratio of luciferase activity measured in U373- X4-TRIM5 cells to that measured in U373-X4-LacZ cells after normalization with results obtained in the same experiment for viruses carrying the NL4-3 CA (Fig. 1). Consistent with prior studies performed using other cell types (27, 32, 65, 68), the infectivities of viruses carrying the NL4-3 CA were 2-fold higher in U373-X4-TRIM5 cells than in U373-X4- LacZ cells. Thus, the infectivity of a virus that is 10 times more sensitive to htrim5 than NL4-3 was approximately 20-fold higher in the U373-X4-TRIM5 cells than in the control cells. We have previously found that the sensitivities to TRIM5 of recombinant viruses NL4-3, NRC1, NRC2, and NRC10 were similar when VSV-pseudotyped viruses and those expressing the HIV-1 envelope were compared (5). Thus, the entry pathway used during infection does not influence this parameter. CTL resistance mutations induce susceptibility to htrim5 in viruses from patient NRC10. We have previously shown that a recombinant virus carrying Gag-PR sequences from plasma TABLE 2. Mutations in capsid common to all viral clones from patient NRC10 a Mutation Epitope HLA type(s) Effect Reference(s), source V159I SV9 B*0702, B*8101 CTL resistance 23, 73 S165N KF11 B*57/5801, B63 CTL resistance 17, 21, 74 S173T KK10 B*27 Compensatory 60, this article KF11 B*57 CTL resistance 48, 53 E203D KA9 B*4002 ND b V218A TW10 B*57/5801, B63 Compensatory 11, 14, 29, 36 M228I TW10 B*57/5801, B63 Compensatory 4, 8, 11, 14, 29, 36, 51 G248A TW10 B*57/5801, B63 CTL resistance 8, 20, 21, 39, 49, 63 R264K KK10 B*27 CTL resistance 25, 33, 60, 61 I267V KK10 B*27 ND L268M KK10 B*27 CTL resistance, compensatory 25, 33, 60, 61 R286K ND S310T QW9 B*5301, B*57/B*5801 CTL resistance 2, 48, 51 G340A ND a For mutations implicated in resistance to CTLs, the CTL epitope involved, its major histocompatibility complex (MHC) restriction, and the effect of the mutation are also shown. b ND, not determined.

4 VOL. 85, 2011 SENSITIVITY OF HIV-1 TO TRIM FIG. 3. Mutations associated with escape from CTL increase sensitivity of viruses from patient NRC10 to htrim5. Recombinant viruses carrying the Gag-PR sequence from the clinical isolate (NRC10) and variants in which one or more mutations in the gag coding sequence were introduced by mutagenesis were prepared. The mutations resulted in the indicated amino acid substitutions in the KK10 epitope and/or Gag amino acid 173 (a) or in the indicated substitutions in the TW10 epitope and/or in or near the CypA binding loop, accompanied or not by reversion of three mutations (S173T, R264K, and L268M) associated with resistance to CTL targeting the KK10 epitope (b). htrim5 sensitivity was determined as shown in Fig. 1. All experiments were performed 4 times using in parallel two independently derived pairs of transduced cell lines (n 8); two different viral stocks were used in the course of the experiments for each series. The infectivity of NL4-3 was fold (a) and fold (b) greater in U373-X4-TRIM5 cells than in U373-X4-LacZ cells., P 0.05;, P 0.01 (compared to NRC10)., P 0.01 compared to NL4-3., P 0.05;, P 0.01;, P (compared to NRC10-T173S-K264R-M268L). viruses obtained from patient NRC10 (whenever a designation such as NRC10 refers to a patient rather than a virus, patient appears before the designation) was more sensitive to htrim5 than a virus expressing Gag-PR sequences from NL4-3 (5). Three different Gag-PR sequences obtained from this patient showed increased sensitivity to htrim5, indicating that this property was shared by the swarm of viruses from this patient and was not due to a rare deleterious point mutation (data not shown). Replacement of the CA coding sequence of this isolate with the corresponding sequence from NL4-3 completely eliminated htrim5 sensitivity, indicating that this phenotype mapped uniquely to CA (data not shown). Patient NRC10 is HLA-B27 positive (Table 1), and the CA sequences of viruses from this patient contained the classical R264K and L268M resistance mutations in the HLA-B27 restricted epitope KK10, as well as the I267V substitution, which has not been implicated in CTL resistance (Fig. 2 and Table 2). Viruses expressing KK10 resistance mutations often also express the S173A compensatory mutation, which alleviates the decrease in replicative capacity resulting from R264K (61). Instead of S173A, viruses from patient NRC10 carried the S173T mutation, which has also been observed in association with KK10 resistance mutations (61). To evaluate the impact of the S173T, R264K, I267V, and L268M mutations on sensitivity to htrim5, one or more of these mutations were removed from the NRC10 sequence by site-directed mutagenesis and the resulting viruses were tested for susceptibility to htrim5 (Fig. 3A). As previously reported (5), NRC10 was 7.2-fold more sensitive to htrim5 than NL4-3. Removing the compensatory S173T mutation alone (NRC10-T173S) increased sensitivity to 16.1 times that of NL4-3. In contrast, removing S173T together with resistance mutation R264K alone (NRC10-T173S-K264R) or together with both R264K and L268M (NRC10-T173S-K264R-M268L) reduced htrim5 sensitivity to 3.5 and 3.7 times that of NL4-3, respectively. Replacing S173T by S173A (NRC10-T173A) also reduced htrim5 sensitivity to 3.6 times that of NL4-3. Reverting the I267V mutation in the NRC10-T173S-K264R-M268L variant (producing NRC10- T173S-K264R-V267I-M268L) had no effect on sensitivity to htrim5. Thus, removing all the mutations implicated in resistance to CTLs targeting KK10 did not completely eliminate the sensitivity of this virus to htrim5. Taken together, these results indicate that (i) the R264K mutation increases the sensitivity of NRC10 to htrim5 and (ii) the impact of R264K can be partially (S173T) or completely (S173A) eliminated by compensatory mutations but that (iii) other mutations independent of those implicated in resistance to CTLs targeting KK10 also contribute to the sensitivity of NRC10 to htrim5. In addition to the four mutations studied above, 9 other amino acids in CA were common to all viral clones obtained from patient NRC10 but differed from those present in NL4-3 (Table 2). Interestingly, 6 of these mutations have been implicated in resistance to CTLs targeting epitopes presented by HLA-B57. Because patient NRC10 is HLA-B57 negative, these mutations are likely to represent footprints selected during prior passage of the virus in an HLA-B57-positive individual (46, 47). Consistent with this idea, the virus from NRC10 did not contain either A163G or T242N, mutations which frequently occur in the KF11 and TW10 epitopes targeted by CTL in HLA-B57-positive individuals but which impair viral replicative capacity and often revert when virus is transmitted to HLA-B57-negative individuals (16, 17, 69). Two of the HLA-B57 footprint mutations found in NRC10 (S165N and S310T in the KF11 and QW9 epitopes) had no

5 11850 BATTIVELLI ET AL. J. VIROL. TABLE 3. Mutations in capsid common to all viral clones from patient NRC2 a Mutation Epitope(s) HLA type(s) Effect Reference(s) A146P IW9, VL10, VV9 B*57/5801, B*15, B*13 CTL resistance 2, 18, 28, 39, 51 A147L IW9, VV9 B*57/5801, B*13 CTL resistance 2, 28, 39, 51 A163G KF11 B*57/5801, B63 15, 17, 21 S165N KF11 B*57/5801, B63 CTL resistance 17, 21, 74 I223V TW10 B*57/5801, B63 compensatory 4, 8, 11, 14, 29, 36, 51 T242N TW10 B*57/5801, B63 CTL resistance 2, 8, 21, 39, 49, 63 G248A TW10 B*57/5801, B63 CTL resistance 2, 8, 20, 21, 39, 49, 63 E260D KK10 B*27 ND b 33, 60 T280V RL9 A2 ND 37 E312D QW9 B*57 CTL resistance? 48, 51, 53 T342S ND B*57, C* , 56 a For mutations implicated in resistance to CTLs, the CTL epitope involved, its MHC restriction, and the effect of the mutation are also shown. b ND, not determined. effect on htrim5 susceptibility (data not shown). In contrast, reversion of the three mutations associated with escape from CTLs targeting the HLA-B57 restricted TW10 epitope (G248A in the TW10 epitope and V218A plus M228I in the cyclophilin A [CypA]-binding loop) led to a substantial decrease in htrim5 sensitivity (Fig. 3b, NRC10-A218V- I228M-A248G). This reduction in htrim5 sensitivity was significantly greater than that observed following reversion of the mutations associated with resistance to the HLA-B27 restricted epitope KK10 (NRC10-T173S-K264R-M268L). Furthermore, reverting the mutations associated with escape from CTLs targeting both the TW10 epitope and the KK10 epitope did not reduce htrim5 sensitivity to a greater extent than reverting only the mutations associated with escape from CTLs targeting the TW10 epitope (Fig. 3b; compare NRC10-A218V- I228M-A248G and NRC10-T173S-K264R-M268L-A218V- I228M-A248G). Among the three mutations associated with escape from CTLs targeting the TW10 epitope, the V218A compensatory mutation had the greatest impact on htrim5 sensitivity. Using the variant in which mutations associated with escape from CTLs targeting the KK10 epitope had been reverted as a starting point (NRC10-T173S-K264R-M268L), reverting V218A alone (NRC10-T173S-K264R-M268L-A218V), reverting both V218A plus M228I (NRC10-T173S-K264R-M268L-A218V- I228M), or reverting all three mutations (NRC10-T173S- K264R-M268L-A218V-I228M-A248G) led to similar reductions in sensitivity to htrim5, whereas reverting only the G248A resistance mutation within the TW10 epitope (NRC10-T173S-K264R-M268L-A248G) did not significantly change htrim5 sensitivity. Taken together, these findings indicate that the relatively high sensitivity of viruses from patient NRC10 to htrim5 required the coexistence of both the V218A compensatory mutation associated with escape from the HLA-B57 restricted epitope TW10 and the R264K resistance mutation associated with escape from the HLA-B27 restricted epitope KK10. In the absence of the V218A mutation, the sensitivity to htrim5 of NRC10 was not influenced by the presence of R264K. Conversely, in the absence of R264K, the impact of the V218A mutation on htrim5 sensitivity was relatively small. When both mutations were present, however, sensitivity to htrim5 was substantially higher and could be further increased by removing the S173T compensatory mutation. Consistent with this interpretation, we found that adding the R264K mutation to NL4-3, which does not carry the V218A mutation, had no significant effect on htrim5 sensitivity (data not shown). CTL resistance mutations induce susceptibility to htrim5 in viruses from patient NRC2. Recombinant viruses carrying Gag-PR sequences of viruses from patient NRC2 are also more sensitive than NL4-3 to htrim5 (5). Patient NRC2 is HLA-B57 positive (Table 1), and viruses from this patient contained the classical T242N and G248A escape mutations in the HLA-B57 restricted epitope TW10 (Table 3). Viruses expressing TW10 resistance mutations often express one or more compensatory mutations in or near the CypA-binding loop, and viruses from patient NRC2 expressed the I223V mutation. To evaluate the impact of these mutations on sensitivity to htrim5, one or more of these mutations were removed from the NRC2 sequence and the sensitivities of viruses carrying these sequences were evaluated (Fig. 4A). Removing the I223V compensatory mutation (NRC2-V223I) did not influence sensitivity to htrim5. Removing all three mutations associated with resistance to CTLs targeting the HLA-B57 restricted epitope TW10 (NRC2-V223I-N242T-A248G) or reversion of I223V plus T242N (NRC2-V223I-N242T) significantly reduced the htrim5 sensitivities of these viruses compared to that of NRC2, whereas reversion of I223V plus G248A (NRC2-V223I-A248G) did not significantly reduce htrim5 sensitivity. Adding two additional TW10-associated compensatory mutations to NRC2 (NRC2 plus H219Q and M228L) had no significant effect on htrim5 sensitivity relative to that of NRC2. These findings indicate that the resistance mutations in the HLA-B57 restricted epitope TW10, especially T242N, explain most of the increased sensitivity of NRC2 to htrim5. In addition to the three mutations studied above, 6 of the 8 other amino acids in the CA from NRC2 that differed from those present in NL4-3 are associated with resistance to CTLs targeting epitopes presented by HLA-B57 (Table 3). Removing the A146P and A147L mutations, involved in resistance to CTLs targeting the IW9 epitope, did not influence the sensitivity of NRC2 to htrim5 (data not shown). The impact of mutations involved in resistance to CTLs targeting the KF11 and QW9 epitopes was not studied in this isolate; the S165N mutation in the KF11 epitope, however, did not influence the sensitivity of NRC10 to htrim5 (data not shown).

6 VOL. 85, 2011 SENSITIVITY OF HIV-1 TO TRIM FIG. 4. Effect of mutations associated with escape from CTL targeting the HLA-B57 associated epitope TW10 on htrim5 sensitivity of viruses from patients NRC2 and NRC1. Recombinant viruses carrying the Gag-PR sequence from the clinical isolate NRC2 (a) or NRC1 (b) and variants in which one or more mutations in the gag coding sequence were introduced by mutagenesis, resulting in the indicated amino acid substitutions in the TW10 epitope and/or substitutions in or near the CypA binding loop were prepared. Sensitivity to htrim5 was evaluated as described for Fig. 1. The experiments were performed 4 times (a) or 3 times (b) using in parallel two independently derived pairs of transduced cell lines (n 8 and n 6, respectively); two different viral stocks were used in the course of the experiments. The infectivity of NL4-3 was fold (a) and fold (b) greater in U373-X4-TRIM5 cells than in U373-X4-LacZ cells., P 0.01;, P (compared to NRC2 [a] or NRC1[b]). The effect of resistance mutations in the TW10 epitope on htrim5 sensitivity is context dependent. Although the T242N and G248A mutations in the TW10 epitope caused a 3-fold increase in the sensitivity of NRC2 viruses to htrim5, the viruses from a third patient, patient NRC1, which express these mutations along with 3 compensatory mutations in the CypA-binding loop, showed little sensitivity to htrim5 (Fig. 4B). As was observed for NRC2, progressive removal from the NRC1 virus of the compensatory mutations in or near the FIG. 5. Relationship between sensitivity to htrim5 and viral infectivity. Sensitivity to htrim5 and viral infectivity, as measured in the target cells in which htrim5 activity had been inhibited by overexpression of TRIM5, were determined as described for Fig. 1 for recombinant viruses carrying the Gag-PR sequence from the clinical isolates (solid symbols) or viral variants produced by mutagenesis (open symbols) whose sensitivity to htrim5 is shown in Fig. 3 and 4. CypA-binding loop did not influence htrim5 sensitivity (NRC1-A218V, NRC1-A218V-Q219H, NRC1-A218V-Q219H- V223I, and NRC1-A218V-Q219H-V223I-L228M). Removing, in addition, the T242N resistance mutation (NRC1-A218V-Q219H- V223I-L228M-N242T) or both T242N and G248A mutations (NRC1-A218V-Q219H-V223I-L228M-N242T-A248G) reduced htrim5 sensitivity to levels that were not significantly different from that for NL4-3. Nevertheless, the presence of the T242N and G248A mutations in NRC1 had substantially less impact on sensitivity to htrim5 sensitivity than was observed for NRC2. Thus, as described earlier for the resistance mutations in the HLA-B27 restricted epitope KK10, the impact of resistance mutations in the HLA-B57 restricted epitope TW10 on htrim5 sensitivity is dependent on variation in the CA sequence outside this epitope. htrim5 sensitivity and viral infectivity. For the mutations studied above, we also examined the relationship between their effects on sensitivity to htrim5 and viral infectivity, as measured in the target cells in which htrim5 activity had been inhibited by overexpression of TRIM5 (Fig. 1). No significant correlations between htrim5 sensitivity and infectivity were observed, either when variants of a given CA sequence were analyzed separately (Fig. 5) (Pearson r: 0.10 to 0.52; P 0.29 for all comparisons) or when all viral variants were analyzed together (Pearson r 0.35; P 0.09). In some cases, changes in htrim5 sensitivity and infectivity were inversely related. For viruses from patient NRC10, in the absence of compensatory mutations at amino acid 173, the R264K and L268M mutations in the HLA-B27 restricted epitope KK10 increased sensitivity to htrim5 (Fig. 3A) and significantly impaired infectivity (Table 4); the presence of either the S173T or S173A compensatory mutation decreased sensitivity to htrim5 and increased infectivity (Table 4) (P 0.02 for both comparisons). In other cases, however, this inverse relationship was not observed. Adding the R264K and L268M mutations to NL4-3 had no significant effect on sensitivity to htrim5 but, as previously described (61), resulted in a 4-fold reduction in infectivity (data not shown). Removing the T242N and G248A resistance mutations in the TW10

7 11852 BATTIVELLI ET AL. J. VIROL. TABLE 4. Viral infectivity, as measured in the target cells in which htrim5 activity had been inhibited by overexpression of TRIM5 Variant Infectivity (% NL4-3) NRC NRC10-T173S NRC10-T173S-K264R NRC10-T173S-K264R-M268L NRC10-T173A NRC10-T173S-K264R-V267I-M268L NRC10-A218V-I228M-A248G NRC10-T173S-K264R-M268L-A218V NRC10-T173S-K264R-M268L-A218V-I228M NRC10-T173S-K264R-M268L-A218V-I228M-A248G NRC10-T173S-K264R-M268L-A248V Mean SD n P a NRC NRC2-V223I NRC2-V223I-A248G NRC2-V223I-N242T NRC2-V223I-N242T-A248G NRC2-H219Q-M228L NRC NRC1-A218V NRC1-A218V-Q219H NRC1-A218V-Q219H-V223I NRC1-A218V-Q219H-V223I-L228M NRC1-A218V-Q219H-V223I-L228M-N242T NRC1-A218V-Q219H-V223I-L228M-N242T-A248G a, P 0.01 compared to NRC10., P 0.02;, P 0.01 (compared to NRC10-T173S)., P 0.01 compared to NRC2;, P 0.01 compared to NRC1. epitope along with compensatory mutations in the CypA-binding loop reduced both htrim5 sensitivity (Fig. 4) and infectivity of NRC2 and NRC1 (Table 4). Thus, no fixed pattern relating changes in htrim5 sensitivity and infectivity was discernible in our studies. DISCUSSION We recently found that HIV-1 expressing CA sequences from some clinical isolates can be more sensitive to htrim5 than are laboratory-adapted strains (5). This study extends these findings by demonstrating that (i) the increased htrim5 sensitivities of these viruses can be attributed to mutations associated with escape from CTL responses against epitopes in CA, (ii) both resistance mutations within CTL epitopes and associated compensatory mutations can contribute to this process, (iii) the impact of these mutations on htrim5 sensitivity is strongly context dependent, in that the effects of a given mutation can be strikingly different depending on the CA sequence in which it occurs, and (iv) CTL resistance mutations and/or associated compensatory mutations previously selected by prior hosts expressing distinct HLA haplotypes appear to influence the impact on htrim5 sensitivity of CTL resistance mutations arising in the recipient. Taken together, these findings suggest that modifications in htrim5 sensitivity resulting from CTL mutations may influence HIV-1 replication in vivo, but, as discussed below, numerous factors are likely to modulate the magnitude of this effect. Several of the resistance mutations studied here are thought to impair viral replication in vivo because the mutations revert when viruses carrying these mutations are transmitted to individuals not expressing HLA molecules capable of presenting the involved epitope. When evaluated in single-cycle infectivity assays, effects of these mutations on infectivity have ranged from a 2-fold reduction (T242N mutation in the B57 restricted TW10 epitope) to a 20-fold reduction (R264K mutation in the B27 restricted KK10 epitope) (8, 44, 61). These effects can be different in different target cells (8, 44), and the possible contribution of htrim5 expression by the target cells was not evaluated. Because we evaluated the effect of htrim5 sensitivity using different target cells and treated these target cells with IFN- to increase htrim5 expression, it is difficult to compare these prior findings with our results. In some cases studied by us, resistance mutations were found to both impair infectivity and increase sensitivity to htrim5, and the addition of compensatory mutations corrected, at least partially, both of these defects. In these cases, the selective pressures to improve infectivity and reduce htrim5 sensitivity would presumably act in consort to favor selection of the compensatory mutations. Further studies are required to evaluate the extent that htrim5 sensitivity influences viral replication in vivo and to assess the relative importance of the effect of CTL mutations on htrim5 sensitivity and viral infectivity. It is likely, however, that the pressure exerted by htrim5 on viral replication by a given host may vary for several reasons. First, we found that the extent that a given CTL resistance mutation influences htrim5 sensitivity is dependent on the CA sequences of viruses infecting that individual. In addition, host TRIM5 activity can be variable depending on the level of expression of htrim5 (64), possibly reflecting polymorphisms in transcription factor-binding sites (10), on

8 VOL. 85, 2011 SENSITIVITY OF HIV-1 TO TRIM the extent of exposure of cells to IFN- (5, 13, 58), and/or on the presence of htrim5 polymorphisms that can modify its ability to inhibit replication of a given viral isolate (5, 24, 30, 59). Furthermore, several of the mutations studied by us, including both the CTL resistance mutations and mutations in the CypA-binding loop, have been previously shown to modulate the effects of inhibiting CA-CypA interactions on viral infectivity (8). The inhibition of CypA-CA interactions often directly reduces the stability of HIV-1 capsid and impairs viral infectivity, although mutations in or near the CypA binding loop can render viruses CypA independent (8, 14, 29, 70). The inhibition of CypA-CA interactions has been reported to have no effect on the sensitivity of HIV-1 to htrim5 (32, 65, 68), but we have recently shown that inhibiting CypA-CA interactions can also increase or decrease htrim5 sensitivity in an isolate-specific fashion (5). These findings suggest that differences in cellular CypA levels may also influence the effect of CTL mutations on both viral replicative capacity and htrim5 sensitivity. Finally, it is conceivable that, for a given virus, escape mutations for certain epitopes targeted by CTL may increase htrim5 sensitivity to such an extent that their emergence is precluded, leaving the virus more sensitive to CTL pressure. ACKNOWLEDGMENTS This work was supported by grants from the Agence Nationale de Recherche sur le Sida et les Hépatites (ANRS) and Sidaction. REFERENCES 1. Aickin, M., and H. Gensler Adjusting for multiple testing when reporting research results: the Bonferroni vs Holm methods. Am. J. Public Health 86: Allen, T. M., et al Selective escape from CD8 T-cell responses represents a major driving force of human immunodeficiency virus type 1 (HIV-1) sequence diversity and reveals constraints on HIV-1 evolution. J. Virol. 79: Allen, T. M., et al Selection, transmission, and reversion of an antigenprocessing cytotoxic T-lymphocyte escape mutation in human immunodeficiency virus type 1 infection. J. Virol. 78: Bailey, J. R., et al Transmission of human immunodeficiency virus type 1 from a patient who developed AIDS to an elite suppressor. J. Virol. 82: Battivelli, E., et al Strain-specific differences in the impact of human TRIM5alpha, different TRIM5alpha alleles, and the inhibition of capsidcyclophilin A interactions on the infectivity of HIV-1. J. Virol. 84: Borghans, J. A., A. Molgaard, R. J. de Boer, and C. Kesmir HLA alleles associated with slow progression to AIDS truly prefer to present HIV-1 p24. PLoS One 2:e Brockman, M. A., et al Early selection in Gag by protective HLA alleles contributes to reduced HIV-1 replication capacity that may be largely compensated for in chronic infection. J. Virol. 84: Brockman, M. A., et al Escape and compensation from early HLA- B57-mediated cytotoxic T-lymphocyte pressure on human immunodeficiency virus type 1 Gag alter capsid interactions with cyclophilin A. J. Virol. 81: Brumme, Z. L., et al HLA-associated immune escape pathways in HIV-1 subtype B Gag, Pol and Nef proteins. PLoS One 4:e Cagliani, R., et al Long-term balancing selection maintains transspecific polymorphisms in the human TRIM5 gene. Hum. Genet. 128: Carlson, J. M., et al Phylogenetic dependency networks: inferring patterns of CTL escape and codon covariation in HIV-1 Gag. PLoS Comput. Biol. 4:e Carrington, M., and S. J. O Brien The influence of HLA genotype on AIDS. Annu. Rev. Med. 54: Carthagena, L., et al Implication of TRIM alpha and TRIMCyp in interferon-induced anti-retroviral restriction activities. Retrovirology 5: Chatterji, U., et al Naturally occurring capsid substitutions render HIV-1 cyclophilin A independent in human cells and TRIM-cyclophilinresistant in owl monkey cells. J. Biol. Chem. 280: Chopera, D. R., et al Transmission of HIV-1 CTL escape variants provides HLA-mismatched recipients with a survival advantage. PLoS Pathog. 4:e Crawford, H., et al Evolution of HLA-B*5703 HIV-1 escape mutations in HLA-B*5703-positive individuals and their transmission recipients. J. Exp. Med. 206: Crawford, H., et al Compensatory mutation partially restores fitness and delays reversion of escape mutation within the immunodominant HLA- B*5703-restricted Gag epitope in chronic human immunodeficiency virus type 1 infection. J. Virol. 81: Draenert, R., et al Immune selection for altered antigen processing leads to cytotoxic T lymphocyte escape in chronic HIV-1 infection. J. Exp. Med. 199: Fitzon, T., et al Proline residues in the HIV-1 NH 2 -terminal capsid domain: structure determinants for proper core assembly and subsequent steps of early replication. Virology 268: Forshey, B. M., U. von Schwedler, W. I. Sundquist, and C. Aiken Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication. J. Virol. 76: Frahm, N., et al HLA-B63 presents HLA-B57/B58-restricted cytotoxic T-lymphocyte epitopes and is associated with low human immunodeficiency virus load. J. Virol. 79: Gamble, T. R., et al Crystal structure of human cyclophilin A bound to the amino-terminal domain of HIV-1 capsid. Cell 87: Goepfert, P. A., et al Transmission of HIV-1 Gag immune escape mutations is associated with reduced viral load in linked recipients. J. Exp. Med. 205: Goldschmidt, V., et al Role of common human TRIM5alpha variants in HIV-1 disease progression. Retrovirology 3: Goulder, P. J., et al Late escape from an immunodominant cytotoxic T-lymphocyte response associated with progression to AIDS. Nat. Med. 3: Goulder, P. J., and D. I. Watkins Impact of MHC class I diversity on immune control of immunodeficiency virus replication. Nat. Rev. Immunol. 8: Hatziioannou, T., D. Perez-Caballero, S. Cowan, and P. D. Bieniasz Cyclophilin interactions with incoming human immunodeficiency virus type 1 capsids with opposing effects on infectivity in human cells. J. Virol. 79: Honeyborne, I., et al Control of human immunodeficiency virus type 1 is associated with HLA-B*13 and targeting of multiple gag-specific CD8 T-cell epitopes. J. Virol. 81: Ikeda, Y., L. M. Ylinen, M. Kahar-Bador, and G. J. Towers Influence of gag on human immunodeficiency virus type 1 species-specific tropism. J. Virol. 78: Javanbakht, H., et al Characterization of TRIM5alpha trimerization and its contribution to human immunodeficiency virus capsid binding. Virology 353: Johnson, W. E., and S. L. Sawyer Molecular evolution of the antiretroviral TRIM5 gene. Immunogenetics 61: Keckesova, Z., L. M. Ylinen, and G. J. Towers Cyclophilin A renders human immunodeficiency virus type 1 sensitive to Old World monkey but not human TRIM5 alpha antiviral activity. J. Virol. 80: Kelleher, A. D., et al Clustered mutations in HIV-1 gag are consistently required for escape from HLA-B27-restricted cytotoxic T lymphocyte responses. J. Exp. Med. 193: Kiepiela, P., et al Dominant influence of HLA-B in mediating the potential co-evolution of HIV and HLA. Nature 432: Kim, J., C. Tipper, and J. Sodroski Role of the TRIM5 RING domain E3 ubiquitin ligase activity in capsid disassembly, reverse transcription blockade and restriction of simian immunodeficiency virus. J. Virol. 85: Kootstra, N. A., M. Navis, C. Beugeling, K. A. van Dort, and H. Schuitemaker The presence of the Trim5alpha escape mutation H87Q in the capsid of late stage HIV-1 variants is preceded by a prolonged asymptomatic infection phase. AIDS 21: Lazaro, E., et al Sequences of clustered epitopes in Gag and Nef potentially presented by predominant class I human leukocyte antigen (HLA) alleles A and B expressed by human immunodeficiency virus type 1 (HIV-1)-infected patients in Vietnam. AIDS Res. Hum. Retroviruses 21: Leslie, A., et al Differential selection pressure exerted on HIV by CTL targeting identical epitopes but restricted by distinct HLA alleles from the same HLA supertype. J. Immunol. 177: Leslie, A. J., et al HIV evolution: CTL escape mutation and reversion after transmission. Nat. Med. 10: Li, B., et al Rapid reversion of sequence polymorphisms dominates early human immunodeficiency virus type 1 evolution. J. Virol. 81: Liang, B., M. Luo, T. B. Ball, S. J. Jones, and F. A. Plummer QUASI analysis of host immune responses to Gag polyproteins of human immunodeficiency virus type 1 by a systematic bioinformatics approach. Biochem. Cell Biol. 88:

9 11854 BATTIVELLI ET AL. J. VIROL. 42. Liu, Y., et al Evolution of human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitopes: fitness-balanced escape. J. Virol. 81: Luban, J Cyclophilin A, TRIM5, and resistance to human immunodeficiency virus type 1 infection. J. Virol. 81: Martinez-Picado, J., et al Fitness cost of escape mutations in p24 Gag in association with control of human immunodeficiency virus type 1. J. Virol. 80: Matsuoka, S., E. Dam, D. Lecossier, F. Clavel, and A. J. Hance Modulation of HIV-1 infectivity and cyclophilin A-dependence by Gag sequence and target cell type. Retrovirology 6: Matthews, P. C., et al HLA footprints on human immunodeficiency virus type 1 are associated with interclade polymorphisms and intraclade phylogenetic clustering. J. Virol. 83: McMichael, A., and P. Klenerman HIV/AIDS. HLA leaves its footprints on HIV. Science 296: Migueles, S. A., et al The differential ability of HLA B*5701 longterm nonprogressors and progressors to restrict human immunodeficiency virus replication is not caused by loss of recognition of autologous viral gag sequences. J. Virol. 77: Miura, T., et al HLA-B57/B*5801 human immunodeficiency virus type 1 elite controllers select for rare gag variants associated with reduced viral replication capacity and strong cytotoxic T-lymphotye [sic] recognition. J. Virol. 83: (Erratum, 83:5961.) 50. Moore, C. B., et al Evidence of HIV-1 adaptation to HLA-restricted immune responses at a population level. Science 296: Navis, M., et al Viral replication capacity as a correlate of HLA B57/B5801-associated nonprogressive HIV-1 infection. J. Immunol. 179: Newman, R. M., and W. E. Johnson A brief history of TRIM5alpha. AIDS Rev. 9: O Connell, K. A., et al Control of HIV-1 in elite suppressors despite ongoing replication and evolution in plasma virus. J. Virol. 84: Pereyra, F., et al The major genetic determinants of HIV-1 control affect HLA class I peptide presentation. International HIV Controllers Study. Science 330: Pertel, T., et al TRIM5 is an innate immune sensor for the retrovirus capsid lattice. Nature 472: Peters, H. O., et al An integrative bioinformatic approach for studying escape mutations in human immunodeficiency virus type 1 gag in the Pumwani sex worker cohort. J. Virol. 82: Price, D. A., et al T cell receptor recognition motifs govern immune escape patterns in acute SIV infection. Immunity 21: Sakuma, R., A. A. Mael, and Y. Ikeda Alpha interferon enhances TRIM5alpha-mediated antiviral activities in human and rhesus monkey cells. J. Virol. 81: Sawyer, S. L., L. I. Wu, J. M. Akey, M. Emerman, and H. S. Malik High-frequency persistence of an impaired allele of the retroviral defense gene TRIM5alpha in humans. Curr. Biol. 16: Schneidewind, A., et al Structural and functional constraints limit options for cytotoxic T-lymphocyte escape in the immunodominant HLA- B27-restricted epitope in human immunodeficiency virus type 1 capsid. J. Virol. 82: Schneidewind, A., et al Escape from the dominant HLA-B27-restricted cytotoxic T-lymphocyte response in Gag is associated with a dramatic reduction in human immunodeficiency virus type 1 replication. J. Virol. 81: Schneidewind, A., et al Transmission and long-term stability of compensated CD8 escape mutations. J. Virol. 83: Schneidewind, A., et al Maternal transmission of human immunodeficiency virus escape mutations subverts HLA-B57 immunodominance but facilitates viral control in the haploidentical infant. J. Virol. 83: Sewram, S., et al Human TRIM5alpha expression levels and reduced susceptibility to HIV-1 infection. J. Infect. Dis. 199: Sokolskaja, E., L. Berthoux, and J. Luban Cyclophilin A and TRIM5alpha independently regulate human immunodeficiency virus type 1 infectivity in human cells. J. Virol. 80: Stremlau, M., et al The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 427: Stremlau, M., et al Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor. Proc. Natl. Acad. Sci. U. S. A. 103: Stremlau, M., B. Song, H. Javanbakht, M. Perron, and J. Sodroski Cyclophilin A: an auxiliary but not necessary cofactor for TRIM5alpha restriction of HIV-1. Virology 351: Thobakgale, C. F., et al Impact of HLA in mother and child on disease progression of pediatric human immunodeficiency virus type 1 infection. J. Virol. 83: Towers, G. J The control of viral infection by tripartite motif proteins and cyclophilin A. Retrovirology 4: Troyer, R. M., et al Variable fitness impact of HIV-1 escape mutations to cytotoxic T lymphocyte (CTL) response. PLoS Pathog. 5:e Virgin, H. W., and B. D. Walker Immunology and the elusive AIDS vaccine. Nature 464: Yu, X. G., et al Consistent patterns in the development and immunodominance of human immunodeficiency virus type 1 (HIV-1)-specific CD8 T-cell responses following acute HIV-1 infection. J. Virol. 76: Yu, X. G., et al Mutually exclusive T-cell receptor induction and differential susceptibility to human immunodeficiency virus type 1 mutational escape associated with a two-amino-acid difference between HLA class I subtypes. J. Virol. 81: