Bertha Cecilia Ramirez

The development of ribosomal profiling (Riboseq) revealed the immense coding capacity of human and viral genomes. Here, we used Riboseq to delineate the translatome of HIV-1 in infected CD4⁺ T cells. In addition to canonical viral protein coding sequences (CDSs), we identify 98 alternative open reading frames (ARFs), corresponding to small Open Reading Frames (sORFs) that are distributed across the HIV genome including the UTR regions. Using a database of HIV genomes, we observe that most ARF amino-acid sequences are likely conserved among clade B and C of HIV-1, with 8 ARF-encoded amino-acid sequences being more conserved than the overlapping CDSs. Using T cell-based assays and mass spectrometry-based immunopeptidomics, we demonstrate that ARFs encode viral polypeptides. In the blood of people living with HIV, ARF-derived peptides elicit potent poly-functional T cell responses mediated by both CD4⁺ and CD8⁺ T cells. Our discovery expands the list of conserved viral polypeptides that are targets for vaccination strategies and might reveal the existence of viral microproteins or pseudogenes.

Human immunodeficiency virus type-1 (HIV-1) is a complex retrovirus that relies on alternative splicing, translational, and post-translational mechanisms to produce over 15 functional proteins from its single ~10 kb transcriptional unit. Using ribosome profiling, nascent protein labeling, RNA sequencing, and whole-proteomics of infected CD4 + T lymphocytes, we characterized the transcriptional, translational, and post-translational landscape during infection. While viral infection exerts a significant impact on host transcript abundance, global translation rates are only modestly affected. Proteomics data reveal extensive transcriptional and post-translational regulation, with many genes showing opposing trends between transcript/ribosome profiling and protein abundance. These findings highlight a complex regulatory network orchestrating gene expression at multiple levels. Viral ribosome profiling further uncovered extensive non-AUG translation of small peptides from upstream open reading frames (uORFs) within the 5’ long terminal repeat, which elicit specific T cell responses in people living with HIV. Conservation of uORF translation among retroviruses, along with TAR sequences, shapes DDX3 dependency for efficient translation of the main viral open reading frames.

CD4 ⁺ T lymphocytes play a major role in the establishment and maintenance of immunity. They are activated by antigenic peptides derived from extracellular or newly synthesized (endogenous) proteins presented by the MHC‐II molecules. The pathways leading to endogenous MHC‐II presentation remain poorly characterized. We demonstrate here that the autophagy receptor, T6BP, influences both autophagy‐dependent and ‐independent endogenous presentation of HIV‐ and HCMV‐derived peptides. By studying the immunopeptidome of MHC‐II molecules, we show that T6BP affects both the quantity and quality of peptides presented. T6BP silencing induces the mislocalization of the MHC‐II‐loading compartments and rapid degradation of the invariant chain (CD74) without altering the expression and internalization kinetics of MHC‐II molecules. Defining the interactome of T6BP, we identify calnexin as a T6BP partner. We show that the calnexin cytosolic tail is required for this interaction. Remarkably, calnexin silencing replicates the functional consequences of T6BP silencing: decreased CD4 ⁺ T cell activation and exacerbated CD74 degradation. Altogether, we unravel T6BP as a key player of the MHC‐II‐restricted endogenous presentation pathway, and we propose one potential mechanism of action.