Background: The HIV-1 5′ leader consists of the RNA upstream of canonical coding regions, encodes essential replicative functions, and exhibits the highest degree of conservation within the viral genome. We hypothesized that the 5′ leader, despite its designation as the 5′ “untranslated region,” encodes previously uncharacterized open reading frames (ORFs) that are expressed from AUG-like start codons and harbor epitopes that are recognized by host T cell responses.
Methods: Single genome sequencing (SGS) was conducted on plasma virus from HIV-1-infected humans. Interferon-γ ELISPOT and intracellular cytokine staining were performed on PBMCs stimulated with autologous peptides or putative escape variants encoded by the 5′ leader sequence. Mass spectrometry analysis of major histocompatibility complex (MHC)-presented epitopes was conducted on peptides purified using MHC immunoprecipitation, acid elution, and reverse phase liquid chromatography of lysates from HIV-IIIB infected CD4+ T cells. Ribosomal profiling was used to identify AUG-like translation initiation sites.
Results: SGS of virus from HIV-1-infected humans demonstrated mutational patterns suggestive of virus escape throughout the 5′ leader. Based on these escape patterns, 4 potential ORFs were identified: one in the transactivation response element (TAR), one within U5, one surrounding the dimerization initiation signal (DIS), and one around the major splice donor. Screening for a T cell response to autologous transmitted/founder and putative escape peptides from these ORFs revealed CD8+ T cell recognition of an epitope within the DIS stem loop ORF. Mass spectrometry analysis of MHC-bound peptides from HIV-IIIB-infected T cells revealed presentation of two overlapping TAR peptides, LA14 and LL8. Ribosomal profiling demonstrated translation initiation at one-off AUGs within these two newly identified ORFs. Analysis of sequences in the Los Alamos HIV compendium showed that 91% of Group M viruses encode a DIS stem loop ORF and 93% of non-Clade A Group M viruses encode a TAR ORF.
Conclusions: We report here the discovery of multiple previously unrecognized ORFs in the HIV-1 5′ leader, or 5′ “untranslated region.” Peptides from these ORFs are presented on infected CD4+ T cells and can target these cells for T cell recognition. The discovery of a novel source of T cell epitopes within such a highly conserved and functionally important region of the genome has implications for both vaccine development and immunopathogenesis research.