Mutations across Murine Hepatitis Virus nsp4 Alter Virus Fitness and Membrane Modifications
Mutations across Murine Hepatitis Virus nsp4 Alter Virus Fitness and Membrane Modifications
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DOI：
10.1128/jvi.02776-14
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发表时间：
2015-02-01
影响因子：
5.4
通讯作者：
Denison, Mark R.
中科院分区：
文献类型：
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作者：
Beachboard, Dia C.;Anderson-Daniels, Jordan M.;Denison, Mark R.
正义RNA病毒感染的一个共同特征是作为病毒RNA合成位点的宿主细胞质膜的修饰。冠状病毒诱导双膜囊泡（DMV），但DMV在复制和病毒适应性中的作用仍不清楚。冠状病毒编码16种非结构蛋白（nsps），其中nsp3、nsp4和nsp6是DMV形成所必需的。先前已经表明，改变nsp4糖基化的鼠肝炎病毒（MHV）nsp4环1中的突变与破坏的DMV形成相关，并导致病毒复制和RNA合成的变化。然而，尚不清楚DMV形态或nsp4糖基化的另一种功能是否对病毒复制产生影响。在这项研究中，我们测试了跨nsp4的突变，无论是单独的还是与消除nsp4糖基化的突变相结合，是否会影响DMV的形成，复制和适应性。与糖基化位点不同的nsp4中的残基，特别是内质网（ER）腔环1中的残基，独立地破坏了DMV的数量和形态，并加剧了与糖基化丧失相关的DMV变化。改变DMV形态但不改变糖基化的突变不影响病毒适应性，而缺乏nsp4糖基化的病毒表现出适应性的丧失。结果支持的假设，DMV的形态和数量不是病毒健身的关键决定因素。结果还表明，nsp4糖基化除了MHV诱导的双膜vesicle.IMPORTANCEAll正义RNA病毒修改病毒复制复合物形成的宿主细胞质膜的组织和稳定性在复制中发挥作用。因此，定义病毒诱导的膜修饰的机制对于理解病毒复制和开发抑制病毒的新方法是必不可少的。冠状病毒引起的膜变化包括双膜囊泡（DMVs）和卷曲膜。已知三种病毒非结构蛋白（nsps）nsp3、nsp4和nsp6是DMV形成所需的。目前尚不清楚这些蛋白质如何诱导膜修饰或蛋白质的哪些区域参与DMV的形成和稳定性。在这项研究中，我们发现nsp4的突变延迟了病毒复制并破坏了DMV的形成，并且nsp4糖基化的丧失与大量的适应性成本有关。这些结果支持nsp4在DMV形成和病毒适应性中的关键作用。
A common feature of infection by positive-sense RNA virus is the modification of host cell cytoplasmic membranes that serve as sites of viral RNA synthesis. Coronaviruses induce double-membrane vesicles (DMVs), but the role of DMVs in replication and virus fitness remains unclear. Coronaviruses encode 16 nonstructural proteins (nsps), three of which, nsp3, nsp4, and nsp6, are necessary and sufficient for DMV formation. It has been shown previously that mutations in murine hepatitis virus (MHV) nsp4 loop 1 that alter nsp4 glycosylation are associated with disrupted DMV formation and result in changes in virus replication and RNA synthesis. However, it is not known whether DMV morphology or another function of nsp4 glycosylation is responsible for effects on virus replication. In this study, we tested whether mutations across nsp4, both alone and in combination with mutations that abolish nsp4 glycosylation, affected DMV formation, replication, and fitness. Residues in nsp4 distinct from glycosylation sites, particularly in the endoplasmic reticulum (ER) luminal loop 1, independently disrupted both the number and morphology of DMVs and exacerbated DMV changes associated with loss of glycosylation. Mutations that altered DMV morphology but not glycosylation did not affect virus fitness while viruses lacking nsp4 glycosylation exhibited a loss in fitness. The results support the hypothesis that DMV morphology and numbers are not key determinants of virus fitness. The results also suggest that nsp4 glycosylation serves roles in replication in addition to the organization and stability of MHV-induced double-membrane vesicles.IMPORTANCEAll positive-sense RNA viruses modify host cytoplasmic membranes for viral replication complex formation. Thus, defining the mechanisms of virus-induced membrane modifications is essential for both understanding virus replication and development of novel approaches to virus inhibition. Coronavirus-induced membrane changes include double-membrane vesicles (DMVs) and convoluted membranes. Three viral nonstructural proteins (nsps), nsp3, nsp4, and nsp6, are known to be required for DMV formation. It is unknown how these proteins induce membrane modification or which regions of the proteins are involved in DMV formation and stability. In this study, we show that mutations across nsp4 delay virus replication and disrupt DMV formation and that loss of nsp4 glycosylation is associated with a substantial fitness cost. These results support a critical role for nsp4 in DMV formation and virus fitness.