High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling

PLoS Pathogens

Stuart G. Siddell¹  Betty Y.-W. Chung²  Andrew E. Firth³  Ian Brierley³  Nerea Irigoyen³  Joshua D. Jones³ 

[1] Department of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom;Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom;Division of Virology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom

关键词: Ribosomes;    RNA viruses;    Messenger RNA;    Protein translation;    Gene prediction;    Negative-sense RNA viruses;    RNA synthesis;    Positive-sense RNA viruses;   

DOI  :  10.1371/journal.ppat.1005473

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus.

【 授权许可】

CC BY