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

Abstract

Certain histocompatibility leukocyte antigen (HLA) alleles are associated with improved clinical outcomes for individuals infected with human immunodeficiency virus type 1 (HIV-1), but the mechanisms for their effects remain undefined. An early CD8(+) T-cell escape mutation in the dominant HLA-B57-restricted Gag epitope TW10 (TSTLQEQIGW) has been shown to impair HIV-1 replication capacity in vitro. We demonstrate here that this T(242)N substitution in the capsid protein is associated with upstream mutations at residues H(219), I(223), and M(228) in the cyclophilin A (CypA)-binding loop in B57(+) individuals with progressive disease. In an independent cohort of epidemiologically linked transmission pairs, the presence of these substitutions in viruses encoding T(242)N was associated with significantly higher plasma viremia in donors, further suggesting that these secondary mutations compensated for the replication defect of T(242)N. Using NL4-3 constructs, we illustrate the ability of these CypA loop changes to partially restore replication of the T(242)N variant in vitro. Notably, these mutations also enhanced viral resistance to the drug cyclosporine A, indicating a reduced dependence of the compensated virus on CypA that is normally essential for optimal infectivity. Therefore, mutations in TW10 allow HIV-1 to evade a dominant early CD8(+) T-cell response, but the benefits of escape are offset by a defect in capsid function. These data suggest that TW10 escape variants undergo a postentry block that is partially overcome by changes in the CypA-binding loop and identify a mechanism for an HIV-1 fitness defect that may contribute to the slower disease progression associated with HLA-B57.

FIG. 1.

Escape in the B57-TW10 epitope is associated with multiple potential compensatory mutations. (a) The predominant HIV-1 Gag sequences from HLA-B57⁺ progressors and nonprogressors are shown, aligned to the consensus subtype B Gag sequence from the LANL HIV database. Patient classification, viral loads (in copies/milliliter), CD4 counts (in cells/microliter), and Gag sequences (accession numbers AV986516 to AV986733) are from Migueles et al. (49). Minor clonal populations are shown in lowercase for Pt5 and Pt12 (asterisks). The HLA-B57-restricted CD8 epitope TW10 (TSTLQEQIGW) is highlighted (boxed), and the CypA-binding domain is indicated (underlined). The predominant TW10 escape mutation, T₂₄₂N, is shaded. Polymorphisms at residues H₂₁₉, I₂₂₃, M₂₂₈, and G₂₄₈ (boldface) were more frequent in HLA-B57⁺ progressors than nonprogressors in this cohort, as well as in subtype B sequences from the LANL HIV database that contained the T₂₄₂N substitution. Statistical calculations were determined using Fisher's exact test, and P values are shown beneath each residue. (b) A structural diagram of the HIV-1 capsid is shown, with residues associated with TW10 escape highlighted. The TW10 epitope is shown (green), and polymorphic residues are identified (red). The escape mutation T₂₄₂N and residues G₂₄₈A and N₂₅₂H are located within or adjacent to alpha helix VI. Residues H₂₁₉Q, I₂₂₃V, and M₂₂₈I are located within the CypA-binding loop.

FIG. 2.

Acquisition of mutations in the CypA-binding loop correlates with higher plasma viremia following transmission of TW10 escape variants. Viral sequence polymorphism data from 22 transmission pairs from a discordant couple cohort in Lusaka, Zambia, were analyzed in which a donor infected with HIV-1 subtype C transmitted the T₂₄₂N escape mutation in the presence of zero, one, two, or three or more potential compensating mutations (i.e., H₂₁₉Q, I₂₂₃V, M₂₂₈I, and G₂₄₈A). (a) In the donors, the median plasma viral load was significantly higher when one or more of these additional mutations was present (P = 0.03). Furthermore, a correlation was observed for donors between viral load and number of mutations (r = 0.34), but this failed to reach significance (P = 0.13). (b) A similar correlation also was observed for recipients between viral load and the number of mutations in this region (r = 0.29), but again this failed to reach statistical significance (P = 0.19). Viral loads were not significantly different with the addition of one or more mutations to the recipients (P = 0.56). (c) When data from donors and recipients were combined for analysis, the median viral load was significantly higher when T₂₄₂N escape occurred in the presence of one or more of the identified mutations (P = 0.03). A correlation was observed between viral load and the number of mutations (r = 0.34), which reached significance with this larger number of samples (P = 0.03).

FIG. 3.

TW10-associated mutations in NL4-3. (a) Variants were aligned to the consensus clade B sequence and were constructed by PCR mutagenesis using the HIV-1 molecular clone NL4-3 as described in Materials and Methods. (b) HEK293T cells were transfected in triplicate with 5 μg of proviral plasmid DNA for each variant as described in Materials and Methods. Culture supernatants were collected at 24 h, and particle production was assessed by p24 ELISA. Values shown are the means ± standard deviations for each variant. No significant differences were observed between WT NL4-3 and the N, S, NA, QNA, or QVINA mutant strain. Replacement of T₂₄₂ with A or W resulted in a severe defect in virion production.

FIG. 4.

Escape in TW10 reduces HIV-1 replication capacity. (a and b) GFP reporter cells were inoculated in quadruplicate cultures with WT or TW10 variant N, S, A, NA, QNA, or QVINA (MOI = 0.0025), and the percentage of GFP⁺ cells was determined by FACS analysis to measure viral spread. Mean values ± standard deviations are shown for the WT, N, and QVINA. The A variant did not replicate efficiently and was included in these experiments as a negative control. (a) In CEM-GXR cells, no substantial differences in replication were seen between the WT (black line) and any of the TW10-associated mutants. (b) In contrast, in Jurkat-GFP cells a significant difference in replication was observed between the WT (black line) and the N variant (red line), as indicated by a 10-fold-greater proportion of GFP⁺ cells in WT-infected cultures at day 5. Incorporation of mutations in the CypA-binding loop enhanced the spread of the N mutant, as seen for the QVINA variant (green line). (c) The natural log slope of cell-to-cell spread in Jurkat-GFP cells was determined for each variant as described in Materials and Methods. Mean values ± standard deviations are shown. Results were compared using Student's t test, and the slope of the WT virus was found to be significantly greater than those of each of the variants (P < 0.001). Furthermore, the QNA and QVINA variants displayed significantly higher slopes than the N mutant (P = 0.039 and 0.003, respectively).

FIG. 5.

Replication of TW10 variants in primary cells. PHA-stimulated PBMCs from a healthy HIV-negative donor were inoculated with TW10 variants at an MOI of 0.001 in the absence (a) or presence (b) of 0.5 μM CsA. Replication of each virus was determined by ELISA to measure p24 in the culture supernatant at the indicated day postinfection, and results are displayed as the mean values of triplicate samples ± standard deviations. (a) In the absence of drug, a reduction in p24 accumulation compared to that of the WT was observed for the N variant (red line) at days 5 and 7 (black line) (P = 0.031 and 0.004, respectively), and replication of the NA variant (orange line) also was reduced on day 7 (P = 0.041). (b) Addition of CsA resulted in a decline in viral replication for all variants. The NA variant (orange line) appeared to be more sensitive to this drug than the WT, while the QVINA strain (green line) was less sensitive to CsA during this 7-day assay.

FIG. 6.

TW10 mutations alter viral sensitivity to CsA. GFP reporter cells were inoculated with VSV-g-pseudotyped WT, N, S, NA, QNA, or QVINA virus (MOI = 0.1) in triplicate in the presence of either 0.5 or 2.5 μM CsA. The proportion of GFP⁺ cells was measured at 48 h, and values were normalized to those of the no-drug control. Mean values ± standard deviations are shown, and significant differences were determined using Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.005). (a) No significant differences were seen between the WT, N, S, and NA variants in CEM-GXR cells, as each appeared to be equally sensitive to both 0.5 and 2.5 μM concentrations of CsA. Infection by the QNA and QVINA variants in the presence of both CsA concentrations was significantly higher than that of the WT (P < 0.01 in all cases), indicating that these variants were resistant to the effects of the drug. (b) In Jurkat-GFP cells, the N, S, and NA variants were significantly more sensitive than WT virus to 0.5 μM CsA (P < 0.001, P < 0.05, and P < 0.001, respectively), while the QVINA variant was significantly more resistant to the drug (P < 0.001). At 2.5 μM CsA, the N mutant was again more sensitive than the WT (P < 0.001), while both the QNA and QVINA variants appeared to be resistant to the drug (P < 0.001 for each).