State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Science, China.
J Virol. 2013 Apr;87(8):4609-22. doi: 10.1128/JVI.02424-12. Epub 2013 Feb 13.
Flavivirus nonstructural protein 2A (NS2A) is a component of the viral replication complex that functions in virion assembly and antagonizes the host immune response. Although flavivirus NS2A is known to associate with the endoplasmic reticulum (ER) membrane, the detailed topology of this protein has not been determined. Here we report the first topology model of flavivirus NS2A on the ER membrane. Using dengue virus (DENV) NS2A as a model, we show that (i) the N-terminal 68 amino acids are located in the ER lumen, with one segment (amino acids 30 to 52) that interacts with ER membrane without traversing the lipid bilayer; (ii) amino acids 69 to 209 form five transmembrane segments, each of which integrally spans the ER membrane; and (iii) the C-terminal tail (amino acids 210 to 218) is located in the cytosol. Nuclear magnetic resonance (NMR) structural analysis showed that the first membrane-spanning segment (amino acids 69 to 93) consists of two helices separated by a "helix breaker." The helix breaker is formed by amino acid P85 and one positively charged residue, R84. Functional analysis using replicon and genome-length RNAs of DENV-2 indicates that P85 is not important for viral replication. However, when R84 was replaced with E, the mutation attenuated both viral RNA synthesis and virus production. Remarkably, an R84A mutation did not affect viral RNA synthesis but blocked intracellular formation of infectious virions. Collectively, the mutagenesis results demonstrate that NS2A functions in both DENV RNA synthesis and virion assembly/maturation. The topology model of DENV NS2A provides a good starting point for studying how flavivirus NS2A modulates viral replication and evasion of host immune response.
黄病毒非结构蛋白 2A（NS2A）是病毒复制复合物的组成部分，在病毒粒子组装和拮抗宿主免疫反应中发挥作用。尽管已知黄病毒 NS2A 与内质网（ER）膜结合，但该蛋白的详细拓扑结构尚未确定。在这里，我们报告了 ER 膜上黄病毒 NS2A 的第一个拓扑模型。我们以登革热病毒（DENV）NS2A 为模型，表明：（i）N 端的 68 个氨基酸位于 ER 腔中，其中一个片段（氨基酸 30 至 52）与 ER 膜相互作用而不穿过脂质双层；（ii）氨基酸 69 至 209 形成五个跨膜段，每个跨膜段完整地横跨 ER 膜；（iii）C 端尾巴（氨基酸 210 至 218）位于细胞质中。核磁共振（NMR）结构分析表明，第一个跨膜段（氨基酸 69 至 93）由两个螺旋组成，它们之间由一个“螺旋破坏者”隔开。螺旋破坏者由氨基酸 P85 和一个带正电荷的残基 R84 组成。使用 DENV-2 的复制子和全长 RNA 的功能分析表明，P85 对病毒复制不重要。然而，当 R84 被替换为 E 时，该突变减弱了病毒 RNA 的合成和病毒的产生。值得注意的是，R84A 突变不会影响病毒 RNA 的合成，但会阻止感染性病毒粒子在细胞内的形成。总的来说，突变分析表明 NS2A 既参与 DENV RNA 的合成，也参与病毒粒子的组装/成熟。DENV NS2A 的拓扑模型为研究黄病毒 NS2A 如何调节病毒复制和逃避宿主免疫反应提供了一个良好的起点。
