Unveiling novel conserved HIV-1 open reading frames encoding T-cell antigens using ribosome profiling.
Ontology highlight
ABSTRACT: Alternative reading frames (ARFs) were described in several viral genomes using ribosome profiling such as HCMV, KSV, and SARS-CoV-2 but no demonstration has been made on HIV genome. Using ribosome profiling, we uncovered 98 conserved HIV ARFs distributed across the genome, with high conservation among HIV clade B and C isolates. Our analysis revealed that at least 42 ARFs encode viral polypeptides, as demonstrated by T-cell responses targeting 45 ARF-derived peptides in patients under treatment or naturally controlling the infection. At the same time, we identified a ligand of HLA-A*0201 derived from an identified ARF on primary infected cells. These responses were mediated by polyfunctional CD4+ T-cells secreting at least 3 cytokines simultaneously. Our discovery expands the list of conserved viral polypeptides that might be potential targets for vaccination strategies.
Project description: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 HIV-infected individuals, 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.
Project description:In Arabidopsis and presumably other flowering plant species as well, there are two non-conserved putative ARF GTPases in addition to the eukaryotically conserved ARF1 (class I) and eight ARF guanine-nucleotide exchange factors (ARF-GEFs) that appear to be involved in specific membrane-trafficking pathways. The aim of the study is to analyze which of the ARFs is activated by which ARF-GEF.
Project description:Plant TRANS-ACTING SIRNA3 (TAS3)-derived short-interfering RNAs (siRNAs) include tasiR-ARFs, which are functionally conserved in targeting AUXIN RESPONSE FACTOR (ARF) genes, and a set of non-tasiR-ARF siRNAs, which have rarely been studied. In this study, TAS3 siRNAs were systematically characterized in rice (Oryza sative). Small RNA-seq results showed that an overwhelming majority of TAS3 siRNAs belong to the non-tasiR-ARF group, while tasiR-ARFs occupy a diminutive fraction. Phylogenetic analysis of TAS3 genes across dicot and monocot plants revealed that the siRNA-generating regions were highly conserved in grass species, especially in the oryzoideae. Target genes were identified for not only tasiR-ARFs but also non-tasiR-ARF siRNAs by analyzing rice degradome datasets, and some of these siRNA-target interactions were experimentally confirmed in rice tas3 mutants. Consistent with altered expression of target genes, phenotypic variations were observed for mutants in three TAS3 loci in comparison to wild-type rice. The regulatory role of ribosomes in the TAS3 siRNA-target interactions was further revealed by the fact that TAS3 siRNA-mediated target cleavage, in particular tasiR-ARFs targeting ARF2/3/14/15, occurred extensively in rice polysome samples. Altogether our study sheds new insights into TAS3 genes in plants and expand our knowledge about rice TAS3 siRNA-target interactions.
Project description:The Epstein-Barr virus (EBV) genome encodes several hundred transcripts. Using total RNA sequencing and ribosome profiling, we have characterized the transcriptional and translational scope of B cells infected with EBV. We could show that viral transcripts are translated at variable efficiency and that several viral genes show ribosome recruitment to the 5’ leader region of mRNAs. We used two different virus strains with differing in vitro characteristics to study EBV translation and could show that in cells infected with the weakly replicating EBV strain some lytic genes showed evidence of monosomal ribosome recruitment mainly in the 5’ leader region and on start codons in the absence of protein production. Finally, we could identify 25 novel upstream open reading frames that potentially regulate the translation efficiency of some viral genes.
Project description:Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). Here, we exploit a systematic CRISPR-based screening strategy to identify hundreds of non-canonical CDSs that are essential for cellular growth and whose disruption elicit specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds that are presented by the HLA system. Interestingly, we find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same mRNA, thus revealing the diverse use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.
Project description:RNA sequencing protocols allow for quantifying gene expression regulation at each individual step, from transcription to protein synthesis. Ribosome Profiling (Ribo-seq) maps the positions of translating ribosomes over the entire transcriptome. Despite its great potential, a rigorous statistical approach to identify translated regions from Ribo-seq data is not yet available. To fill this gap, we developed RiboTaper, which quantifies the significance of the three-nucleotide periodicity of Ribo-seq reads via spectral analysis methods. Examination of RNA fragments protected by ribosome
Project description:Dicer enzymes function at the core of RNA silencing to defend against exogenous RNA, or as an endogenous mechanism of gene regulation. Plant DICER-LIKE4 (DCL4) performs dual functions, acting in antiviral defense, as well as in development via the biogenesis of tasiR-ARFs. These small RNAs play an essential role in the grasses and act to spatially define the expression domain of AUXIN RESPONSE FACTOR3 (ARF3) transcription factors. However, contrary to tasiR-ARFs’ essential function in development, DCL4 proteins exhibit strong evidence of recurrent adaptation typical of host factors involved in antiviral immunity. Here, we address how DCL4 balances its role in development with pressures to diversify in response to viral attack. We show that, in contrast to other tasiR-ARF biogenesis mutants, dcl4 null alleles condition an uncharacteristically mild phenotype, correlated with normal expression of select arf3 targets. Loss of DCL4 activity yields a class of 22-nt tasiR-ARF variants associated with the processing of arf3 transcripts into 22-nt secondary siRNAs by DCL1. Our findings uncover the presence of a novel DCL1-dependent siRNA pathway that bypasses the otherwise adverse developmental effects of DCL4 mutations. This novel pathway is predicted to have important implications for DCL4’s role in antiviral defense by reducing the selective constraints on DCL4 and allowing it to diversify in response to viral suppressors. Examination of small RNAs isolated from dcl4, dcl1 and double mutants in imbibed kernels of maize
Project description:Accurate annotations of protein coding regions are essential for understanding how genetic information is translated into biological functions. The recent development of ribosome footprint profiling provides an important new tool for measuring translation. Here we describe riboHMM, a new method that uses ribosome footprint data along with gene expression and sequence information to accurately infer translated sequences. We applied our method to human lymphoblastoid cell lines and identified 7,863 previously unannotated coding sequences, including 445 translated sequences in pseudogenes and 2,442 translated upstream open reading frames. We observed an enrichment of harringtonine-treated ribosome footprints at the inferred initiation sites, validating many of the novel coding sequences. In aggregate, the novel sequences exhibit significant signatures of purifying selection indicative of protein-coding function, suggesting that many of the novel sequences are functional. We observed that nearly 40% of bicistronic transcripts showed significant negative correlation in the levels of translation of their two coding sequences, suggesting a key regulatory role for these novel translated sequences. Despite evidence for their functional importance, the novel peptide sequences were detected by mass spectrometry at a lower rate than predicted based on data from annotated proteins, thus suggesting that many of the novel peptide products may be relatively short-lived. Our work illustrates the value of ribosome profiling for improving coding annotations, and significantly expands the set of known coding regions.
Project description:Translational control is a key determinant of protein abundance, which in turns defines the physiology and pathology of human cells. Initiation of translation is highly regulated in eukaryotes and is considered as the rate-limiting step of protein synthesis. mRNA secondary structures in 5’ untranslated region (UTR) and associated helicases have been characterised as key determinants of translation initiation. Nevertheless the transcriptome-wide contribution of non-canonical secondary structures, such as RNA G-quadruplexes (rG4s), to the translation of human mRNAs remains largely unappreciated. Here we use a ribosome profiling strategy to investigate the translational landscape associated to rG4s-containing mRNAs and the contribution of two rG4s-specialised DExH-box helicases, DHX9 and DHX36, to translation initiation in human cells. We show that rG4-forming sequences in 5’-UTR is associated with decreased translation efficiency which correlate with an increased ribosome density within the 5’-UTRs. We found that rG4s contribute to the translation of upstream open reading frames, and as a consequence, thwart the translation of the associated protein coding sequences (CDS). Depletion of the DHX36 and DHX9 helicases demonstrated that the formation of the rG4 structural motif rather than its nucleotide sequence mediate translation initiation. Our findings unveil a role for non-canonical structures in defining alternative 5’ starts for human mRNAs translation initiation.