Partially Uncleaved Alphavirus Replicase Forms Spherule Structures in the Presence and Absence of RNA Template
Partially Uncleaved Alphavirus Replicase Forms Spherule Structures in the Presence and Absence of RNA Template
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DOI：
10.1128/jvi.00787-17
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发表时间：
2017-09-01
影响因子：
5.4
通讯作者：
Ahola, Tero
中科院分区：
文献类型：
--
作者：
Hellstrom, Kirsi;Kallio, Katri;Ahola, Tero
甲病毒是正链RNA病毒，其复制酶表达为多聚蛋白P1234，其被切割成四种最终产物，非结构蛋白nsP1至nsP4。复制酶蛋白与病毒RNA和宿主因子一起形成称为小球的膜内陷，其作为复制复合物产生子代RNA。我们以前已经表明，野生型甲病毒复制酶需要一个功能性的RNA模板和活性聚合酶产生小球结构。然而，我们现在发现，在没有RNA或复制的情况下，复制酶蛋白质的特定部分加工形式可以单独引起膜内陷。小球体形成的最低要求是表达正确切割的nsP4，以及未切割的P123或nsP1和未切割的P23的组合。这些不活跃的小球形态不太规则比复制诱导的小球。在模板的存在下，nsP1加上未切割的P23加上nsP4可以有效地组装活性复制小球，产生负义和正义RNA链。单独的P23不具有膜亲和力，但可以在nsP1和nsP4存在下被募集到膜位点。这些结果定义了一组所需的病毒成分的甲病毒复制复合物的组装，并建议的可能性，它可以从单独表达的nonstructural proteins.IMPORTANCE重建所有正链RNA病毒广泛修改宿主细胞膜作为有效的平台，病毒RNA复制。甲病毒和其他几个组诱导保护膜内陷（小球）作为其基因组工厂。大多数正链病毒产生其复制酶作为多蛋白前体，其通过精确和受调控的裂解进一步加工。我们在这里表明，甲病毒复制酶的特异性切割中间体可以在没有病毒RNA的情况下产生小球结构。在模板RNA的存在下，相同的中间体产生活性复制复合物。因此，部分裂解的复制酶蛋白在连接复制复合物组装、膜变形和RNA合成的不同阶段中起着关键作用。
Alphaviruses are positive-strand RNA viruses expressing their replicase as a polyprotein, P1234, which is cleaved to four final products, nonstructural proteins nsP1 to nsP4. The replicase proteins together with viral RNA and host factors form membrane invaginations termed spherules, which act as the replication complexes producing progeny RNAs. We have previously shown that the wild-type alphavirus replicase requires a functional RNA template and active polymerase to generate spherule structures. However, we now find that specific partially processed forms of the replicase proteins alone can give rise to membrane invaginations in the absence of RNA or replication. The minimal requirement for spherule formation was the expression of properly cleaved nsP4, together with either uncleaved P123 or with the combination of nsP1 and uncleaved P23. These inactive spherules were morphologically less regular than replication-induced spherules. In the presence of template, nsP1 plus uncleaved P23 plus nsP4 could efficiently assemble active replication spherules producing both negative-sense and positive-sense RNA strands. P23 alone did not have membrane affinity, but could be recruited to membrane sites in the presence of nsP1 and nsP4. These results define the set of viral components required for alphavirus replication complex assembly and suggest the possibility that it could be reconstituted from separately expressed nonstructural proteins.IMPORTANCE All positive-strand RNA viruses extensively modify host cell membranes to serve as efficient platforms for viral RNA replication. Alphaviruses and several other groups induce protective membrane invaginations (spherules) as their genome factories. Most positive-strand viruses produce their replicase as a polyprotein precursor, which is further processed through precise and regulated cleavages. We show here that specific cleavage intermediates of the alphavirus replicase can give rise to spherule structures in the absence of viral RNA. In the presence of template RNA, the same intermediates yield active replication complexes. Thus, partially cleaved replicase proteins play key roles that connect replication complex assembly, membrane deformation, and the different stages of RNA synthesis.