SARS-CoV-2 ORF6 Disrupts Bidirectional Nucleocytoplasmic Transport through Interactions with Rae1 and Nup98.
SARS-CoV-2 ORF6 Disrupts Bidirectional Nucleocytoplasmic Transport through Interactions with Rae1 and Nup98.
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DOI：
10.1128/mbio.00065-21
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发表时间：
2021-04-13
期刊：
影响因子：
6.4
通讯作者：
Greninger AL
中科院分区：
文献类型：
--
作者：
Addetia A;Lieberman NAP;Phung Q;Hsiang TY;Xie H;Roychoudhury P;Shrestha L;Loprieno MA;Huang ML;Gale M Jr;Jerome KR;Greninger AL
SARS-CoV-2是2019冠状病毒病（COVID-19）的病原体，是一种RNA病毒，其基因组大，可编码多种辅助蛋白。虽然这些辅助蛋白不是体外生长所必需的，但它们可以促进病毒的致病性。在细胞质中复制的RNA病毒通常会破坏核细胞质转运，以优先翻译自己的转录本并阻止宿主的抗病毒反应。sarbecvirus附属蛋白ORF6先前已被证明是严重急性呼吸综合征冠状病毒（SARS-CoV）和严重急性呼吸综合征冠状病毒2 （SARS-CoV-2）中干扰素产生的主要抑制剂。在这里，我们发现与模拟感染的细胞相比，sars - cov -2感染的细胞显示出更高水平的核mRNA积累。我们证明ORF6负责宿主mRNA的核监禁，并使用共转染报告基因实验，我们发现mRNA的核保留阻断了新转录mRNA的表达。ORF6对宿主mRNA的核包裹与其与mRNA输出因子Rae1和Nup98的共化能力有关。这些蛋白-蛋白相互作用映射到ORF6的C端，并且可以通过Met58中的单个氨基酸突变来消除。在SARS-CoV-2 ORF6存在时，过表达Rae1可恢复报告基因的表达。SARS-CoV ORF6也与Rae1和Nup98相互作用。然而，与SARS-CoV-2 ORF6相比，SARS-CoV-2 ORF6与Rae1和Nup98的结合更强烈，导致报告蛋白的表达显著降低，这是与SARS-CoV-2大流行独特相关的延迟症状发作和症状前传播的潜在机制。我们还发现SARS-CoV和SARS-CoV-2 ORF6都能阻断多种宿主蛋白的核输入。总之，这些数据支持ORF6通过与Rae1和Nup98的相互作用堵塞核孔以阻止核输入和输出的模型，使宿主细胞无法对SARS-CoV-2感染做出反应。
SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), is an RNA virus with a large genome that encodes multiple accessory proteins. While these accessory proteins are not required for growth in vitro, they can contribute to the pathogenicity of the virus. RNA viruses that replicate in the cytoplasm often disrupt nucleocytoplasmic transport to preferentially translate their own transcripts and prevent host antiviral responses. The Sarbecovirus accessory protein ORF6 has previously been shown to be a major inhibitor of interferon production in both severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we show SARS-CoV-2-infected cells display an elevated level of nuclear mRNA accumulation compared to mock-infected cells. We demonstrate that ORF6 is responsible for this nuclear imprisonment of host mRNA, and using a cotransfected reporter assay, we show this nuclear retention of mRNA blocks expression of newly transcribed mRNAs. ORF6’s nuclear entrapment of host mRNA is associated with its ability to copurify with the mRNA export factors, Rae1 and Nup98. These protein-protein interactions map to the C terminus of ORF6 and can be abolished by a single amino acid mutation in Met58. Overexpression of Rae1 restores reporter expression in the presence of SARS-CoV-2 ORF6. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly copurifies with Rae1 and Nup98 and results in significantly reduced expression of reporter proteins compared to SARS-CoV ORF6, a potential mechanism for the delayed symptom onset and presymptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. We also show that both SARS-CoV and SARS-CoV-2 ORF6 block nuclear import of a broad range of host proteins. Together, these data support a model in which ORF6 clogs the nuclear pore through its interactions with Rae1 and Nup98 to prevent both nuclear import and export, rendering host cells incapable of responding to SARS-CoV-2 infection.