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Ribosome profiling of the retrovirus murine leukemia virus


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Abstract

Background The retrovirus murine leukemia virus (MuLV) has an 8.3 kb RNA genome with a simple 5′-gag-pol-env-3′ architecture. Translation of the pol gene is dependent upon readthrough of the gag UAG stop codon; whereas the env gene is translated from spliced mRNA transcripts. Here, we report the first high resolution analysis of retrovirus gene expression through tandem ribosome profiling (RiboSeq) and RNA sequencing (RNASeq) of MuLV-infected cells. Results Ribosome profiling of MuLV-infected cells was performed, using the translational inhibitors harringtonine and cycloheximide to distinguish initiating and elongating ribosomes, respectively. Meta-analyses of host cell gene expression demonstrated that the RiboSeq datasets specifically captured the footprints of translating ribosomes at high resolution. Direct measurement of ribosomal occupancy of the MuLV genomic RNA indicated that ~7% of ribosomes undergo gag stop codon readthrough to access the pol gene. Initiation of translation was found to occur at several additional sites within the 5′ leaders of the gag and env transcripts, upstream of their respective annotated start codons. Conclusions These experiments reveal the existence of a number of previously uncharacterised, ribosomally occupied open reading frames within the MuLV genome, with possible regulatory consequences. In addition, we provide the first direct measurements of stop codon readthrough efficiency during cellular infection.

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Keywords

Retrovirus, Ribosome profiling, RNASeq, Viral translation,, Murine leukemia virus, Upstream ORF

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Publisher

BioMed Central
Sponsorship
Wellcome Trust (106207/Z/14/Z)
European Research Council (646891)
Biotechnology and Biological Sciences Research Council (BB/G020272/1)
Wellcome Trust (092334/Z/10/Z)
Medical Research Council (MR/M011747/1)
Biotechnology and Biological Sciences Research Council (BB/L000334/1)
Sir Henry Wellcome Postdoctoral Fellowship (Wellcome Trust) to N.I.; Wellcome Trust grant [106207] and European Research Council (ERC) grant [646891] to A.E.F.; U.K. Biotechnology and Biological Research Council (BBSRC) grant [BB/G020272/1] and U.K. Medical Research Council (MRC) grant [MR/M011747/1] to I.B. The funders played no role in the study, or writing of the manuscript.