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Abstract：
Betacoronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), are important pathogens causing potentially lethal infections in humans and animals. Coronavirus RNA synthesis is thought to be associated with replication organelles (ROs) consisting of modified endoplasmic reticulum (ER) membranes. These are transformed into double-membrane vesicles (DMVs) containing viral double-stranded RNA and into other membranous elements such as convoluted membranes, together forming a reticulovesicular network. Previous evidence suggested that the nonstructural proteins (nsp\'s) 3, 4, and 6 of the severe acute respiratory syndrome coronavirus (SARS-CoV), which contain transmembrane domains, would all be required for DMV formation. We have now expressed MERS-CoV replicase self-cleaving polyprotein fragments encompassing nsp3-4 or nsp3-6, as well as coexpressed nsp3 and nsp4 of either MERS-CoV or SARS-CoV, to characterize the membrane structures induced. Using electron tomography, we demonstrate that for both MERS-CoV and SARS-CoV coexpression of nsp3 and nsp4 is required and sufficient to induce DMVs. Coexpression of MERS-CoV nsp3 and nsp4 either as individual proteins or as a self-cleaving nsp3-4 precursor resulted in very similar DMVs, and in both setups we observed proliferation of zippered ER that appeared to wrap into nascent DMVs. Moreover, when inactivating nsp3-4 polyprotein cleavage by mutagenesis, we established that cleavage of the nsp3/nsp4 junction is essential for MERS-CoV DMV formation. Addition of the third MERS-CoV transmembrane protein, nsp6, did not noticeably affect DMV formation. These findings provide important insight into the biogenesis of coronavirus DMVs, establish strong similarities with other nidoviruses (specifically, the arteriviruses), and highlight possible general principles in viral DMV formation.IMPORTANCE The RNA replication of positive stranded RNA viruses of eukaryotes is thought to take place at cytoplasmic membranous replication organelles (ROs). Double-membrane vesicles are a prominent type of viral ROs. They are induced by coronaviruses, such as SARS-CoV and MERS-CoV, as well as by a number of other important pathogens, yet little is known about their biogenesis. In this study, we explored the viral protein requirements for the formation of MERS-CoV- and SARS-CoV-induced DMVs and established that coexpression of two of the three transmembrane subunits of the coronavirus replicase polyprotein, nonstructural proteins (nsp\'s) 3 and 4, is required and sufficient to induce DMV formation. Moreover, release of nsp3 and nsp4 from the polyprotein by proteolytic maturation is essential for this process. These findings provide a strong basis for further research on the biogenesis and functionality of coronavirus ROs and may point to more general principles of viral DMV formation.
摘要：
Betacoronavirus,例如中东呼吸综合征冠状病毒（MERS-CoV），是导致人类和动物潜在致命感染的重要病原体。冠状病毒RNA合成被认为与由修饰的内质网（ER）膜组成的复制细胞器（RO）有关。这些被转化为含有病毒双链RNA的双膜囊泡（DMV）和其他膜元件，如卷积膜，一起形成网状泡状网络。先前的证据表明，严重急性呼吸综合征冠状病毒（SARS-CoV）的非结构蛋白（nsp/s）3、4和6，包含跨膜结构域，都需要DMV的形成。我们现在已经表达了MERS-CoV复制酶自切割多蛋白片段，包括nsp3-4或nsp3-6，以及共表达的MERS-CoV或SARS-CoV的nsp3和nsp4，表征诱导的膜结构。用电子层析成像,我们证明，对于MERS-CoV和SARS-CoV，nsp3和nsp4的共表达是必需的，并且足以诱导DMV。MERS-CoVnsp3和nsp4作为单个蛋白质或作为自切割nsp3-4前体的共表达导致非常相似的DMV，在这两种设置中，我们观察到拉链式ER的扩散，似乎包裹在新生的DMV中。此外，当通过诱变灭活nsp3-4多蛋白裂解时，我们确定nsp3/nsp4结的裂解对于MERS-CoVDMV的形成至关重要。添加第三个MERS-CoV跨膜蛋白，nsp6没有明显影响DMV的形成。这些发现为冠状病毒DMV的生物发生提供了重要的见解，与其他净病毒建立了很强的相似性(特别是，动脉炎病毒)，并强调病毒DMV形成的可能的一般原则。重要性真核生物正链RNA病毒的RNA复制被认为发生在细胞质膜复制细胞器(RO)。双膜囊泡是一种突出的病毒RO类型。它们是由冠状病毒诱导的，如SARS-CoV和MERS-CoV，以及许多其他重要的病原体，然而对它们的生物发生知之甚少。在这项研究中,我们探索了形成MERS-CoV和SARS-CoV诱导的DMV的病毒蛋白需求，并确定了冠状病毒复制酶多蛋白的三个跨膜亚基中的两个的共表达，非结构蛋白（nsp）3和4是必需的，并且足以诱导DMV形成。此外，通过蛋白水解成熟从多蛋白中释放nsp3和nsp4对于该过程至关重要。这些发现为进一步研究冠状病毒RO的生物发生和功能提供了坚实的基础，并可能指出病毒DMV形成的更一般原理。
