As a small retrovirus, HIV-1 depends on the host cell machinery to support its life cycle. Host kinases can phosphorylate HIV-1, which can influence functional states of viral proteins and may influence viral replicative fitness. How HIV-1 phosphorylation affects viral immune recognition by cytotoxic T cells is unknown at present. Notably, recognition of phosphorylated epitopes by CD8 T cells is well documented in the context of lymphoma and melanoma.
In vitro infected CD4 T cells (n=4) and cell-free HIV-1 isolated from culture supernatants (n=3) were subjected to LC-MS/MS mass spectrometry. Selected identified phosphorylated HIV-1 peptides were synthesized and tested for HLA class I binding. PBMCs from HIV-1 infected patients were stimulated with phosphorylated and non-phosphorylated peptides, followed by flow-cytometric analysis of antigen-specific effector response (Fig A, B).
We identified 30 unique and largely novel site-specific phosphorylation in HIV-1, the majority of which were located in the Gag, Pol, and Rev proteins. Of 74 optimal CTL epitopes within Gag, 34 contained identified phosphorylated sites, suggesting that phosphorylation may influence immune recognition by CTLs. Out of six CTL epitopes with detectable phospho-residues selected for further analysis, three phosphorylated epitopes were recognized by CTLs from HIV-1 infected patients in direct ex-vivo assessments. Although CTL responses to these phosphorylated peptides were polyfunctional, the magnitude and cross reactivity of these phospho epitope-specific CTL varied with both epitope and HLA background (Fig A, B). Specifically, while both B*5701 and B*5703 patients responded to phosphorylated version of the immunodominant KF11 epitope, these responses were lower in magnitude and functional avidity when compared with the non-phosphorylated KF11 (Fig A). Cloning of KF11-specific CTLs indicated that cross-reactivity between the phosphorylated and the corresponding non-phosphorylated epitope were mediated by single TCRs (Fig C).
Host kinases can phosphorylate HIV-1 at immunogenic regions. Multiple phosphorylated CTL epitopes can be recognized by HIV-1-specific CTLs, and TCRs able to cross-recognize phosphorylated epitopes are naturally recruited, suggesting that presentation of phosphorylated epitopes may occur in vivo. Phospho-epitope specific T cell responses may modulate the efficacy of antiviral cellular immune responses.