Department of Biochemistry & Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA.
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China.
mBio. 2019 Oct 29;10(5):e02375-19. doi: 10.1128/mBio.02375-19.
The flavivirus virion consists of an envelope outer layer, formed by envelope (E) and membrane (M) proteins on a lipid bilayer, and an internal core, formed by capsid (C) protein and genomic RNA. The molecular mechanism of flavivirus assembly is not well understood. Here, we show that Zika virus (ZIKV) NS2A protein recruits genomic RNA, the structural protein prM/E complex, and the NS2B/NS3 protease complex to the virion assembly site and orchestrates virus morphogenesis. Coimmunoprecipitation analysis showed that ZIKV NS2A binds to prM, E, NS2B, and NS3 (but not C, NS4B, or NS5) in a viral RNA-independent manner, whereas prM/E complex does not interact with NS2B/NS3 complex. Remarkably, a single-amino-acid mutation (E103A) of NS2A impairs its binding to prM/E and NS2B/NS3 and abolishes virus production, demonstrating the indispensable role of NS2A/prM/E and NS2A/NS2B/NS3 interactions in virion assembly. In addition, RNA-protein pulldown analysis identified a stem-loop RNA from the 3' untranslated region (UTR) of the viral genome as an "RNA recruitment signal" for ZIKV assembly. The 3' UTR RNA binds to a cytoplasmic loop of NS2A protein. Mutations of two positively charged residues (R96A and R102A) from the cytoplasmic loop reduce NS2A binding to viral RNA, leading to a complete loss of virion assembly. Collectively, our results support a virion assembly model in which NS2A recruits viral NS2B/NS3 protease and structural C-prM-E polyprotein to the virion assembly site; once the C-prM-E polyprotein has been processed, NS2A presents viral RNA to the structural proteins for virion assembly. ZIKV is a recently emerged mosquito-borne flavivirus that can cause devastating congenital Zika syndrome in pregnant women and Guillain-Barré syndrome in adults. The molecular mechanism of ZIKV virion assembly is largely unknown. Here, we report that ZIKV NS2A plays a central role in recruiting viral RNA, structural protein prM/E, and viral NS2B/NS3 protease to the virion assembly site and orchestrating virion morphogenesis. One mutation that impairs these interactions does not significantly affect viral RNA replication but selectively abolishes virion assembly, demonstrating the specific role of these interactions in virus morphogenesis. We also show that the 3' UTR of ZIKV RNA may serve as a "recruitment signal" through binding to NS2A to enter the virion assembly site. Following a coordinated cleavage of C-prM-E at the virion assembly site, NS2A may present the viral RNA to C protein for nucleocapsid formation followed by envelopment with prM/E proteins. The results have provided new insights into flavivirus virion assembly.
黄病毒病毒粒子由一个包膜外层和一个内部核心组成，包膜外层由包膜 (E) 和膜 (M) 蛋白组成，位于一个脂质双层上，内部核心由衣壳 (C) 蛋白和基因组 RNA 组成。黄病毒装配的分子机制尚不清楚。在这里，我们表明 Zika 病毒 (ZIKV) NS2A 蛋白将基因组 RNA、结构蛋白 prM/E 复合物和 NS2B/NS3 蛋白酶复合物招募到病毒装配部位，并协调病毒形态发生。共免疫沉淀分析表明，ZIKV NS2A 以病毒 RNA 非依赖性方式与 prM、E、NS2B 和 NS3（但不与 C、NS4B 或 NS5）结合，而 prM/E 复合物不与 NS2B/NS3 复合物相互作用。值得注意的是，NS2A 中的单个氨基酸突变 (E103A) 损害了其与 prM/E 和 NS2B/NS3 的结合，并完全抑制了病毒的产生，这表明 NS2A/prM/E 和 NS2A/NS2B/NS3 相互作用在病毒粒子装配中不可或缺。此外，RNA-蛋白下拉分析鉴定出病毒基因组 3' 非翻译区（UTR）中的一个茎环 RNA 作为 ZIKV 装配的“RNA 募集信号”。3'UTR RNA 与 NS2A 蛋白的一个细胞质环结合。来自细胞质环的两个正电荷残基 (R96A 和 R102A) 的突变减少了 NS2A 与病毒 RNA 的结合，导致完全丧失病毒粒子装配。总的来说，我们的结果支持一种病毒粒子装配模型，其中 NS2A 招募病毒 NS2B/NS3 蛋白酶和结构 C-prM-E 多蛋白到病毒粒子装配部位；一旦 C-prM-E 多蛋白被加工，NS2A 就会将病毒 RNA 呈递给结构蛋白以进行病毒粒子装配。ZIKV 是一种新近出现的蚊媒黄病毒，可导致孕妇严重的 Zika 综合征和成人吉兰-巴雷综合征。ZIKV 病毒粒子的装配机制在很大程度上尚不清楚。在这里，我们报告 ZIKV NS2A 在招募病毒 RNA、结构蛋白 prM/E 和病毒 NS2B/NS3 蛋白酶到病毒粒子装配部位以及协调病毒形态发生方面发挥核心作用。一种损害这些相互作用的突变不会显著影响病毒 RNA 复制，但选择性地完全抑制病毒粒子装配，表明这些相互作用在病毒形态发生中具有特异性。我们还表明，ZIKV RNA 的 3'UTR 可能通过与 NS2A 结合作为“募集信号”进入病毒粒子装配部位。在病毒粒子装配部位 C-prM-E 的协调切割之后，NS2A 可能将病毒 RNA 呈递给 C 蛋白以形成核衣壳，然后用 prM/E 蛋白进行包膜。结果为黄病毒病毒粒子装配提供了新的见解。
