1Division of Molecular Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
2Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
J Gen Virol. 2018 Oct;99(10):1391-1406. doi: 10.1099/jgv.0.001134. Epub 2018 Aug 10.
Virus-host interactions play important roles in virus infection and host cellular response. Several viruses, including dengue virus (DENV), usurp host chaperones to support their amplification and survival in the host cell. We investigated the interaction of nonstructural protein 1 (NS1) of DENV with three endoplasmic reticulum-resident chaperones (i.e. GRP78, calnexin and calreticulin) to delineate their functional roles and potential binding sites for protein complex formation. GRP78 protein showed prominent association with DENV NS1 in virus-infected Huh7 cells as evidenced by co-localization and co-immunoprecipitation assays. Further studies on the functional interaction of GRP78 protein were performed by using siRNA-mediated gene knockdown in a DENV replicon transfection system. GRP78 knockdown significantly decreased intracellular NS1 production and delayed NS1 secretion but had no effect on viral RNA replication. Dissecting the important domain of GRP78 required for DENV NS1 interaction showed co-immunoprecipitation of DENV NS1 with a full-length and substrate-binding domain (SBD), but not an ATPase domain, of GRP78, confirming their interaction through SBD binding. Molecular dynamics simulations of DENV NS1 and human GRP78 complex revealed their potential binding sites through hydrogen and hydrophobic bonding. The majority of GRP78-binding sites were located in a β-roll domain and connector subdomains on the DENV NS1 structure involved in hydrophobic surface formation. Taken together, our findings demonstrated the roles of human GRP78 in facilitating the intracellular production and secretion of DENV NS1 as well as predicted potential binding sites between the DENV NS1 and GRP78 complex, which could have implications in the future development of target-based antiviral drugs.
病毒-宿主相互作用在病毒感染和宿主细胞反应中起着重要作用。包括登革热病毒（DENV）在内的几种病毒会篡夺宿主伴侣蛋白，以支持其在宿主细胞中的扩增和存活。我们研究了 DENV 的非结构蛋白 1（NS1）与三种内质网驻留伴侣蛋白（即 GRP78、钙网蛋白和钙调蛋白）的相互作用，以描绘它们的功能作用和潜在的蛋白复合物形成结合位点。共定位和共免疫沉淀实验证实，GRP78 蛋白与感染 DENV 的 Huh7 细胞中的 DENV NS1 有明显的关联。在 DENV 复制子转染系统中，通过 siRNA 介导的基因敲低进一步研究了 GRP78 蛋白的功能相互作用。GRP78 敲低显著降低了细胞内 NS1 的产生并延迟了 NS1 的分泌，但对病毒 RNA 复制没有影响。分析 GRP78 与 DENV NS1 相互作用所必需的重要结构域显示，DENV NS1 与全长和底物结合结构域（SBD）而不是 ATP 酶结构域的 GRP78 发生共免疫沉淀，证实它们通过 SBD 结合相互作用。DENV NS1 和人源 GRP78 复合物的分子动力学模拟揭示了它们通过氢键和疏水键结合的潜在结合位点。大多数 GRP78 结合位点位于 DENV NS1 结构的β-折叠结构域和连接器亚结构域上，这些结构域参与形成疏水面。综上所述，我们的研究结果表明，人源 GRP78 可促进 DENV NS1 的细胞内产生和分泌，并预测了 DENV NS1 和 GRP78 复合物之间的潜在结合位点，这可能对基于靶标的抗病毒药物的未来开发具有重要意义。
