Abstract
BackgroundSARS-CoV-2 is a recently emerged respiratory pathogen that has significantly impacted global human health. We wanted to rapidly characterise the transcriptomic, proteomic and phosphoproteomic landscape of this novel coronavirus to provide a fundamental description of the virus’s genomic and proteomic potential.MethodsWe used direct RNA sequencing to determine the transcriptome of SARS-CoV-2 grown in Vero E6 cells which is widely used to propagate the novel coronavirus. The viral transcriptome was analysed using a recently developed ORF-centric pipeline. Allied to this, we used tandem mass spectrometry to investigate the proteome and phosphoproteome of the same virally infected cells.ResultsOur integrated analysis revealed that the viral transcripts (i.e. subgenomic mRNAs) generally fitted the expected transcription model for coronaviruses. Importantly, a 24 nt in-frame deletion was detected in over half of the subgenomic mRNAs encoding the spike (S) glycoprotein and was predicted to remove a proposed furin cleavage site from the S glycoprotein. Tandem mass spectrometry identified over 500 viral peptides and 44 phosphopeptides in virus-infected cells, covering almost all proteins predicted to be encoded by the SARS-CoV-2 genome, including peptides unique to the deleted variant of the S glycoprotein.ConclusionsDetection of an apparently viable deletion in the furin cleavage site of the S glycoprotein, a leading vaccine target, shows that this and other regions of SARS-CoV-2 proteins may readily mutate. The furin site directs cleavage of the S glycoprotein into functional subunits during virus entry or exit and likely contributes strongly to the pathogenesis and zoonosis of this virus. Our data emphasises that the viral genome sequence should be carefully monitored during the growth of viral stocks for research, animal challenge models and, potentially, in clinical samples. Such variations may result in different levels of virulence, morbidity and mortality.
Highlights
SARS-CoV-2 is a recently emerged respiratory pathogen that has significantly impacted global human health
During viral genome replication a set of “nested” subgenomic mRNAs are produced that are predicted to encode the structural proteins spike (S), envelope (E), membrane (M) and nucleocapsid (N) and at least nine small accessory proteins, some of which are unique to SARS-CoV-2 [3, 4]
There is a need for independent confirmation that viral transcripts are expressed, which can be achieved by tandem mass spectrometry-based proteomics
Summary
SARS-CoV-2 is a recently emerged respiratory pathogen that has significantly impacted global human health. Since the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) as a human pathogen at the end of 2019, the virus has spread globally, causing almost 11.1 million confirmed cases of COVID19 and over half a million deaths as of the 7 July 2020 [1]. Based on homology to other known coronaviruses [3, 4], the genome sequence was used for viral transcript prediction and the annotation of ORFs. Coronaviruses use a complex strategy to express their genetic information [5, 6], involving a process of discontinuous transcription during minus-strand RNA synthesis that is regulated by defined transcription regulatory sequences (TRS) [7]. The subgenomic mRNAs have a common 5′ leader sequence and are 3′ co-terminal with a polyA tail derived from the viral genome
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