Deciphering Poxvirus Gene Expression by RNA Sequencing and Ribosome Profiling

Abstract

The more than 200 closely spaced annotated open reading frames, extensive transcriptional read-through, and numerous unpredicted RNA start sites have made the analysis of vaccinia virus gene expression challenging. Genome-wide ribosome profiling provided an unprecedented assessment of poxvirus gene expression. By 4 h after infection, approximately 80% of the ribosomeassociated mRNA was viral. Ribosome-associated mRNAs were detected for most annotated early genes at 2 h and for most intermediate and late genes at 4 and 8 h. Cluster analysis identified a subset of early mRNAs that continued to be translated at the later times. At 2 h, there was excellent correlation between the abundance of individual mRNAs and the numbers of associated ribosomes, indicating that expression was primarily transcriptionally regulated. However, extensive transcriptional readthrough invalidated similar correlations at later times. The mRNAs with the highest density of ribosomes had host response, DNA replication, and transcription roles at early times and were virion components at late times. Translation inhibitors were used to map initiation sites at single-nucleotide resolution at the start of most annotated open reading frames although in some cases a downstream methionine was used instead. Additional putative translational initiation sites with AUG or alternative codons occurred mostly within open reading frames, and fewer occurred in untranslated leader sequences, antisense strands, and intergenic regions. However, most open reading frames associated with these additional translation initiation sites were short, raising questions regarding their biological roles. The data were used to construct a high-resolution genome-wide map of the vaccinia virus translatome. Poxviruses comprise a large family that infect vertebrates and invertebrates, including species that are highly pathogenic for humans as well as for wild and domesticated animals (1, 2). Nevertheless, attenuated poxviruses are being employed as recombinant vectors for vaccines and potential cancer therapeutics. The cytoplasmic site of replication, the large linear double-stranded DNA genome with covalently closed ends, and the encoding of most, if not all, proteins required for DNA and RNA synthesis are distinguishing features of poxviruses. Studies with poxviruses have led to fundamental and far-reaching discoveries, such as the structure and biosynthesis of the 5= cap (3, 4) and the 3= poly(A) sequence (5, 6) on mRNA and numerous proteins that counteract host defenses (7,8). Vaccinia virus (VACV), the prototype poxvirus that was used as the vaccine to eradicate smallpox, has a 200-kbp genome with more than 200 annotated open reading frames (ORFs) that are selectively expressed at early, intermediate, or late times of infection due to stage-specific viral promoters and transcription factors that work in concert with the multisubunit viral DNA-dependent RNA polymerase. Recent genome-wide transcription analyses (high-throughput sequencing of RNA tran-

The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis

“…Profiling has proven to be increasingly valuable in studies of the translation process, for example, in the discovery of novel open reading frames (ORFs), the determination of elongation rates, the identification of sites of ribosome pausing and in the study of protein folding (for review, see Morris 2009;Weiss and Atkins 2011;Michel and Baranov 2013;Ingolia 2014;Jackson and Standart 2015). It also has broad application in the analysis of global gene expression and has been exploited in studies of infectious diseases (Stern-Ginossar et al 2012, 2015Liu et al 2013;Arias et al 2014;Caro et al 2014;Jensen et al 2014;Muzzey et al 2014;Vasquez et al 2014;Yang et al 2015), cell growth, differentiation and development Huang et al 2013;Lee et al 2013;Stadler and Fire, 2013;Stumpf et al 2013;Subramaniam et al 2013;Baudin-Baillieu et al 2014;Brubaker et al 2014;Duncan and Mata 2014;Gonzalez et al 2014;Hendriks et al 2014;Katz et al 2014;Kronja et al 2014;Schrader et al 2014;Vaidyanathan et al 2014;de Klerk et al 2015), apoptosis (Wiita et al 2013), mitochondrial gene expression and disease (Rooijers et al 2013;Williams et al 2014), cell stress (Gerashenko et al 2012;Labunskyy et al 2014;Zid and O'Shea 2014;Sidrauski et al 2015), cell toxicity …”

Le profilage ribosomique

“…Il est aussi possible d'étudier les mécanismes de traduction des ARNm viraux, déjà décrits par le passé mais dont les détails moléculaires restaient mal définis, en suivant la localisation des ribosomes. Le profilage ribosomique ouvre également de nouvelles perspectives pour l'étude du cycle de réplication de virus particulière-ment complexes, tels que les poxvirus [29] ou encore le cytomégalovi-rus humain (HCMV) [17], grâce à la découverte de nouvelles protéines virales et potentiellement de mécanismes de traduction des ARNm viraux encore jamais décrits. Cette approche est d'autre part très prometteuse pour l'étude des interactions entre le virus et la cellule-hôte en permettant de suivre l'impact d'une infection virale sur la synthèse protéique cellulaire [42].…”

“…C'est le cas notamment pour le virus leucémogène murin (MuLV) qui utilise un codon CUG situé en amont du codon canonique pour produire une isoforme de sa polyprotéine Gag qui n'est pas incorporée dans les particules virales, mais qui joue un rôle important dans la dissémination des virions [27,28]. Le profilage ribosomique appliqué à l'étude de la traduction au cours d'une infection virale a également permis récemment d'identifier de nombreux sites d'initiation non-canoniques chez deux virus à ADN et un virus à ARN [17,24,29]. La plupart des sites d'initiation non-AUG découverts à ce jour chez les virus sont utilisés pour la synthèse de petits peptides dont le rôle biologique n'est pas encore connu.…”

Molluscum Contagiosum Virus Transcriptome in Abortively Infected Cultured Cells and a Human Skin Lesion

“…The determination of RNA start sites and ribosome profiling, as has been done for VACV (31,46), would provide a more precise transcription map. RNA-seq and the reporter gene assays described here may be useful for testing other cell lines and conditions that ultimately could provide an in vitro replication system.…”