CTL Escape Mediated by Proteasomal Destruction of an HIV-1 CrypticEpitope

摘要

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5Dor Q9VF/5N peptides were almost equal indicating a possible cross-reactivity ofthe same CTLs on the two peptides. We then dissected the cellular mechanismsinvolved in the presentation of Q9VF variants. As expected, cells infected withHIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. Incontrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor byQ9VF/5D-specific CTLs. Using in vitro proteasomal digestionsand MS/MS analysis, we demonstrate that the 5N variation introduces a strongproteasomal cleavage site within the epitope, leading to a dramatic reduction ofQ9VF epitope production. Our results strongly suggest that HIV-1 escapes CTLsurveillance by introducing mutations leading to HIV ARF-epitope destruction byproteasomes.