Martin Marie-France, Bonaventure Boris, McCray Nia E, Peersen Olve B, Rozen-Gagnon Kathryn, Stapleford Kenneth A
Department of Microbiology, New York University Grossman School of Medicine, New York, New York, USA.
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
bioRxiv. 2024 Jan 15:2024.01.15.575630. doi: 10.1101/2024.01.15.575630.
Alphaviruses encode an error-prone RNA-dependent RNA polymerase (RdRp), nsP4, required for genome synthesis, yet how the RdRp functions in the complete alphavirus life cycle is not well-defined. Previous work using chikungunya virus (CHIKV) has established the importance of the nsP4 residue cysteine 483 in maintaining viral genetic fidelity. Given the location of residue C483 in the nsP4 palm domain, we hypothesized that other residues within this domain and surrounding subdomains would also contribute to polymerase function. To test this hypothesis, we designed a panel of nsP4 variants via homology modeling based on the Coxsackievirus B3 3 polymerase. We rescued each variant in both mammalian and mosquito cells and discovered that the palm domain and ring finger subdomain contribute to polymerase host-specific replication and genetic stability. Surprisingly, in mosquito cells, these variants in the ring finger and palm domain were replication competent and produced viral structural proteins, but they were unable to produce infectious progeny, indicating a yet uncharacterized role for the polymerase in viral assembly. Finally, we have identified additional residues in the nsP4 palm domain that influence the genetic diversity of the viral progeny, potentially via an alteration in NTP binding and/or discrimination by the polymerase. Taken together, these studies highlight that distinct nsP4 subdomains regulate multiple processes of the alphavirus life cycle, placing nsP4 in a central role during the switch from RNA synthesis to packaging and assembly.
甲病毒编码一种易出错的依赖RNA的RNA聚合酶（RdRp），即nsP4，它是基因组合成所必需的，但RdRp在完整的甲病毒生命周期中的功能尚不清楚。此前使用基孔肯雅病毒（CHIKV）的研究已经证实了nsP4残基半胱氨酸483在维持病毒遗传保真度方面的重要性。鉴于C483残基在nsP4掌状结构域中的位置，我们推测该结构域及其周围子结构域中的其他残基也会对聚合酶功能有贡献。为了验证这一假设，我们基于柯萨奇病毒B3 3聚合酶通过同源建模设计了一组nsP4变体。我们在哺乳动物细胞和蚊子细胞中拯救了每个变体，发现掌状结构域和无名指子结构域有助于聚合酶宿主特异性复制和遗传稳定性。令人惊讶的是，在蚊子细胞中，无名指和掌状结构域中的这些变体具有复制能力并产生病毒结构蛋白，但它们无法产生有感染性的后代，这表明聚合酶在病毒组装中具有尚未明确的作用。最后，我们在nsP4掌状结构域中鉴定出了其他影响病毒后代遗传多样性的残基，可能是通过聚合酶对NTP结合和/或识别的改变来实现的。综上所述，这些研究突出表明，不同的nsP4子结构域调节甲病毒生命周期的多个过程，使nsP4在从RNA合成到包装和组装的转换过程中发挥核心作用。
