Evidence of differential HLA class I-mediated viral evolution in functional and accessory/regulatory genes of HIV-1

Abstract

Despite the formidable mutational capacity and sequence diversity of HIV-1, evidence suggests that viral evolution in response to specific selective pressures follows generally predictable mutational pathways. Population-based analyses of clinically derived HIV sequences may be used to identify immune escape mutations in viral genes; however, prior attempts to identify such mutations have been complicated by the inability to discriminate active immune selection from virus founder effects. Furthermore, the association between mutations arising under in vivo immune selection and disease progression for highly variable pathogens such as HIV-1 remains incompletely understood. We applied a viral lineage-corrected analytical method to investigate HLA class I-associated sequence imprinting in HIV protease, reverse transcriptase (RT), Vpr, and Nef in a large cohort of chronically infected, antiretrovirally naïve individuals. A total of 478 unique HLA-associated polymorphisms were observed and organized into a series of "escape maps," which identify known and putative cytotoxic T lymphocyte (CTL) epitopes under selection pressure in vivo. Our data indicate that pathways to immune escape are predictable based on host HLA class I profile, and that epitope anchor residues are not the preferred sites of CTL escape. Results reveal differential contributions of immune imprinting to viral gene diversity, with Nef exhibiting far greater evidence for HLA class I-mediated selection compared to other genes. Moreover, these data reveal a significant, dose-dependent inverse correlation between HLA-associated polymorphisms and HIV disease stage as estimated by CD4(+) T cell count. Identification of specific sites and patterns of HLA-associated polymorphisms across HIV protease, RT, Vpr, and Nef illuminates regions of the genes encoding these products under active immune selection pressure in vivo. The high density of HLA-associated polymorphisms in Nef compared to other genes investigated indicates differential HLA class I-driven evolution in different viral genes. The relationship between HLA class I-associated polymorphisms and lower CD4(+) cell count suggests that immune escape correlates with disease status, supporting an essential role of maintenance of effective CTL responses in immune control of HIV-1. The design of preventative and therapeutic CTL-based vaccine approaches could incorporate information on predictable escape pathways.

Figure 1. Differential Density of HLA Class I-Associated Viral Polymorphisms across HIV Protease, Reverse Transcriptase, Vpr, and Nef

Polymorphism maps display the percentage amino acid variation over protease (A), reverse transcriptase (B), Vpr (C), and Nef (D). Red × symbols indicate codons at which at least one significant HLA-associated polymorphism was observed with q < 0.2.

Figure 2. Epitope Support Maps: HLA-Associated Polymorphisms Mapping within or Directly Proximal to Published HLA-Restricted CTL Epitopes in Protease/Reverse Transcriptase and Vpr

HLA-associated polymorphisms occurring within or directly proximal (± 3 aa) to published HLA-restricted CTL epitopes in protease/RT and Vpr are shown. Polymorphisms associated with the same HLA allele(s) occurring within, or directly proximal to (± 3 aa) published HLA-restricted epitope boundaries are boxed together in yellow. The published epitope sequence (and associated HLA) is indicated above the epitope boundary arrow. If associations within the same epitope boundaries are ascribed to several linked HLA alleles, the HLA that is known to present the epitope is indicated next to it. Red (“escape”) amino acids indicate positive correlations (amino acids that are enriched in the presence of a specific HLA allele). Thus, “escape” (red) amino acids presumably reflect the escape variant specific for that HLA allele. Blue (“reversion”) amino acids indicate negative correlations, where the presence of a specific HLA allele is associated with the absence of a particular amino acid at a specific position—or, likewise, where the absence of the specific HLA allele is associated with the presence of this amino acid. Thus, “reversion” (blue) amino acids presumably reflect the immunologically susceptible form specific for that HLA allele, and the amino acid to which the sequence may revert upon transmission to an HLA-unmatched host. Underlined associations indicate potential HLA anchor residue sites. Proximal associations (within ± 3 aa of epitope boundaries) are marked with an asterisk (*). Associations additionally supported by functional IFN-γ (ELISpot) data are indicated in green.

Figure 3. Epitope Support Map: HLA-Associated Polymorphisms Mapping within or Directly Proximal to Published HLA-Restricted CTL Epitopes in Nef

HLA-associated polymorphisms occurring within or directly proximal (± 3 aa) to published HLA-restricted CTL epitopes in Nef are shown. Polymorphisms associated with the same HLA allele(s) occurring within, or directly proximal to (± 3 aa) published HLA-restricted epitope boundaries are boxed together in yellow. The published epitope sequence (and associated HLA) is indicated above the epitope boundary arrow. If associations within the same epitope boundaries are ascribed to several linked HLA alleles, the HLA that is known to present the epitope is indicated next to it. Red (“escape”) amino acids indicate positive correlations (amino acids that are enriched in the presence of a specific HLA allele). Thus, “escape” (red) amino acids presumably reflect the escape variant specific for that HLA allele. Blue (“reversion”) amino acids indicate negative correlations, where the presence of a specific HLA allele is associated with the absence of a particular amino acid at a specific position—or, likewise, where the absence of the specific HLA allele is associated with the presence of this amino acid. Thus, “reversion” (blue) amino acids presumably reflect the immunologically susceptible form specific for that HLA allele, and the amino acid to which the sequence may revert upon transmission to an individual lacking that HLA allele. Underlined associations indicate potential HLA anchor residue sites. Proximal associations (within ± 3 aa of epitope boundaries) are marked with an asterisk (*). Associations additionally supported by functional IFN-γ (ELISpot) data are indicated in green

Figure 4. Motif Support Maps: HLA-Associated Polymorphisms Mapping within or Directly Proximal to HLA-Restricted CTL Epitope Motifs in Protease/Reverse Transcriptase and Vpr

HLA-associated polymorphisms occurring within or directly proximal to (± 3 aa) known HLA-restricted epitope anchor residue motifs in protease/RT and Vpr are shown. Polymorphisms associated with the same HLA allele(s) occurring within or directly proximal to (± 3 aa) of HLA-restricted epitope anchor residue motifs are boxed together in yellow. Predicted epitopes (where available) are indicated above the epitope boundary arrow, while HLA-restricted epitope motif boundaries are simply marked by their corresponding HLA association. Where multiple overlapping motifs were present, only the most commonly observed one (based on analysis of individual sequences) is indicated. Linked alleles are indicated in brackets, and in ranked order based on p-value of the association. Red (“escape”) amino acids indicate positive correlations (amino acids that are enriched in the presence of a specific HLA allele thus presumably represent escape variants). Blue (“reversion”) amino acids indicate negative correlations, where the presence of a specific HLA allele is associated with the absence of a particular amino acid at a specific position—and vice versa—in this case, the amino acid presumably reflects the immunologically susceptible form specific for that HLA allele. Underlined associations indicate potential HLA anchor residue sites. Proximal associations (within ± 3 aa of epitope boundaries) are marked with an asterisk (*). Associations supported by functional IFN-γ (ELISpot) data are indicated in green. Where an association is supported solely by ELISpot data, but no predicted epitope or motif, the arrow and lines are all green.

Figure 5. Motif Support Maps: HLA-Associated Polymorphisms Mapping within or Directly Proximal to HLA-Restricted CTL Epitope Motifs in Nef

HLA-associated polymorphisms occurring within or directly proximal to (± 3 aa) known HLA-restricted epitope anchor residue motifs in Nef are shown. Polymorphisms associated with the same HLA allele(s) occurring within or directly proximal to (± 3 aa) of HLA-restricted epitope anchor residue motifs are boxed together in yellow. Predicted epitopes (where available) are indicated above the epitope boundary arrow, while HLA-restricted epitope motif boundaries are simply marked by their corresponding HLA association. Where multiple overlapping motifs were present, only the most commonly observed one (based on analysis of individual sequences) is indicated. Linked alleles are indicated in brackets, and in ranked order based on p-value of the association. Red (“escape”) amino acids indicate positive correlations (amino acids that are enriched in the presence of a specific HLA allele thus presumably represent escape variants). Red dashes indicate positions where the presence of the HLA is associated with deletion of the amino acid at that position. Blue (“reversion”) amino acids indicate negative correlations, where the presence of a specific HLA allele is associated with the absence of a particular amino acid at a specific position—and vice versa—in this case, the amino acid presumably reflects the immunologically susceptible form specific for that HLA allele. Underlined associations indicate potential HLA anchor residue sites. Proximal associations (within ± 3 aa of epitope boundaries) are marked with an asterisk (*). Associations supported by functional IFN-γ (ELISpot) data are indicated in green. Where an association is supported solely by ELISpot data, but no predicted epitope or motif, the arrow and lines are all green.

Figure 6. No Support Maps: HLA-Associated Polymorphisms in Reverse Transcriptase, Vpr, and Nef That Do Not Map within Known or Putative Epitopes, and Are Not Supported by Functional IFN-γ (ELISpot) Data

Associations displayed on this map are likely to be highly enriched for the expected proportion of false-positives (20% with q < 0.2). However, we cannot rule out the possibility that these may represent processing escape mutations occurring distant from the epitope site, compensatory mutations, unusual epitopes, or other factors. Note that no “unsupported” HLA-associated polymorphisms were observed in HIV protease.

Figure 7. Inverse Dose-Response Relationship between Accumulation of Epitope-Associated Escape Mutations in Protease/Reverse Transcriptase, Vpr, and Nef and CD4⁺ Cell Count in Chronic Untreated HIV Infection

A significant inverse dose-response relationship is observed between the presence of epitope-associated escape mutations in protease/RT (A), Vpr (B), and Nef (C) and CD4⁺ cell count in chronic untreated HIV infection. Escape mutations were defined as all significant HLA/HIV associations which mapped within or proximal to (± 3 aa) known CTL-restricted epitopes (listed in Figures 2 and 3). Analysis was restricted to participants able to exhibit “escape,” i.e., those possessing at least one HLA allele featured in Figures 2 and 3 (for each gene investigated). Linked alleles were removed to avoid double counting of associations. Diamonds and bars represent median and interquartile ranges, respectively, of pretherapy CD4⁺ cell count for each of the associated escape strata. Overall p-value obtained using the Kruskal-Wallis test. Significant (p < 0.05) pairwise associations are indicated by brackets.