Characterisation of the transcriptome and proteome of SARS-CoV-2 using direct RNA sequencing and tandem mass spectrometry reveals evidence for a cell passage induced in-frame deletion in the spike glycoprotein that removes the furin-like cleavage site
Characterisation of the transcriptome and proteome of SARS-CoV-2 using direct RNA sequencing and tandem mass spectrometry reveals evidence for a cell passage induced in-frame deletion in the spike glycoprotein that removes the furin-like cleavage site
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DOI：
10.1101/2020.03.22.002204
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发表时间：
2020-03
期刊：
影响因子：
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通讯作者：
A. Davidson;M. Williamson;Sebastian Lewis;D. Shoemark;M. Carroll;K. Heesom;M. Zambon;J. Ellis;Phillip A. Lewis;J. Hiscox;D. Matthews
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文献类型：
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作者：
A. Davidson;M. Williamson;Sebastian Lewis;D. Shoemark;M. Carroll;K. Heesom;M. Zambon;J. Ellis;Phillip A. Lewis;J. Hiscox;D. Matthews
在Vero E6细胞中生长的SARS-CoV-2转录组的特征是使用牛津纳米孔小分子的直接RNA测序。该细胞系正被广泛用于繁殖新型冠状病毒。使用最近开发的以ORF为中心的管道对病毒转录组进行分析。这揭示了病毒转录物(即亚基因组mRNAs)的模式，总体上符合冠状病毒的预测复制和转录模型。在编码SPEKE(S)糖蛋白的亚基因组mRNAs中检测到24nT的框内缺失。这一功能在超过一半的作图转录本中被发现，并被预测将从S糖蛋白中移除一个拟议的呋喃裂解位点。这个基序引导S糖蛋白在病毒进入或退出过程中被切割成功能亚基。S糖蛋白的裂解可以成为人畜共患病冠状病毒传播的屏障，影响病毒的致病性。与此转录组分析相结合，串联质谱仪被用于鉴定500多个病毒多肽和44个磷酸多肽，几乎覆盖了SARS-CoV-2基因组预测的所有编码蛋白，包括S糖蛋白缺失变体特有的多肽。在S糖蛋白的Furin裂解位点检测到一个明显可行的缺失，加强了这一点，即该蛋白和SARS-CoV-2蛋白的其他区域很容易发生突变。鉴于人们对S糖蛋白作为潜在疫苗靶点的兴趣，以及观察到Furin裂解位点可能与该病毒的发病和人畜共患病密切相关，这一点具有明显的意义。在用于研究、动物挑战模型和潜在临床样本的病毒库的增长过程中，应仔细监测病毒基因组序列。这种变异可能会导致不同程度的毒力、发病率和死亡率。
Direct RNA sequencing using an Oxford Nanopore MinION characterised the transcriptome of SARS-CoV-2 grown in Vero E6 cells. This cell line is being widely used to propagate the novel coronavirus. The viral transcriptome was analysed using a recently developed ORF-centric pipeline. This revealed the pattern of viral transcripts, (i.e. subgenomic mRNAs), generally fitted the predicted replication and transcription model for coronaviruses. A 24 nt in-frame deletion was detected in subgenomic mRNAs encoding the spike (S) glycoprotein. This feature was identified in over half of the mapped transcripts and was predicted to remove a proposed furin cleavage site from the S glycoprotein. This motif directs cleavage of the S glycoprotein into functional subunits during virus entry or exit. Cleavage of the S glycoprotein can be a barrier to zoonotic coronavirus transmission and affect viral pathogenicity. Allied to this transcriptome analysis, tandem mass spectrometry was used to identify over 500 viral peptides and 44 phosphopeptides, covering almost all of the proteins predicted to be encoded by the SARS-CoV-2 genome, including peptides unique to the deleted variant of the S glycoprotein. Detection of an apparently viable deletion in the furin cleavage site of the S glycoprotein reinforces the point that this and other regions of SARS-CoV-2 proteins may readily mutate. This is of clear significance given the interest in the S glycoprotein as a potential vaccine target and the observation that the furin cleavage site likely contributes strongly to the pathogenesis and zoonosis of this virus. 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.