Molecular Virology Laboratory, VA-MD College of Veterinary Medicine and Maryland Pathogen Research Institute, University of Maryland, College Park, Maryland, USA.
Liver Diseases Branch, National Institute of Diabetes and Digestive Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
J Virol. 2018 Apr 13;92(9). doi: 10.1128/JVI.00072-18. Print 2018 May 1.
Movement of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC). Karyopherins comprise a family of soluble transport factors facilitating the nucleocytoplasmic translocation of proteins through the NPC. In this study, we found that karyopherin α6 (KPNA6; also known as importin α7) was required for the optimal replication of porcine reproductive and respiratory syndrome virus (PRRSV) and Zika virus (ZIKV), which are positive-sense, single-stranded RNA viruses replicating in the cytoplasm. The KPNA6 protein level in virus-infected cells was much higher than that in mock-infected controls, whereas the KPNA6 transcript remains stable. Viral infection blocked the ubiquitin-proteasomal degradation of KPNA6, which led to an extension of the KPNA6 half-life and the elevation of the KPNA6 level in comparison to mock-infected cells. PRRSV nsp12 protein induced KPNA6 stabilization. KPNA6 silencing was detrimental to the replication of PRRSV, and KPNA6 knockout impaired ZIKV replication. Moreover, KPNA6 knockout blocked the nuclear translocation of PRRSV nsp1β but had a minimal effect on two other PRRSV proteins with nuclear localization. Exogenous restitution of KPNA6 expression in the KPNA6-knockout cells results in restoration of the nuclear translocation of PRRSV nsp1β and the replication of ZIKV. These results indicate that KPNA6 is an important cellular factor for the replication of PRRSV and ZIKV. Positive-sense, single-stranded RNA (+ssRNA) viruses replicate in the cytoplasm of infected cells. The roles of transport factors in the nucleocytoplasmic trafficking system for the replication of +ssRNA viruses are not known. In this study, we discovered that PRRSV and ZIKV viruses needed karyopherin α6 (KPNA6), one of the transport factors, to enhance the virus replication. Our data showed that viral infection induced an elevation of the KPNA6 protein level due to an extension of the KPNA6 half-life via viral interference of the ubiquitin-proteasomal degradation of KPNA6. Notably, KPNA6 silencing or knockout dramatically reduced the replication of PRRSV and ZIKV. PRRSV nsp1β depended on KPNA6 to translocate into the nucleus. In addition, exogenous restitution of KPNA6 expression in KPNA6-knockout cells led to the restoration of nsp1β nuclear translocation and ZIKV replication. These results reveal a new aspect in the virus-cell interaction and may facilitate the development of novel antiviral therapeutics.
大分子在细胞质和细胞核之间的运动是通过核孔复合物（NPC）进行的。核输入蛋白家族是一组可溶性运输因子，可促进蛋白质通过 NPC 的核质转运。在这项研究中，我们发现核输入蛋白 α6（KPNA6；也称为导入素 α7）对于猪繁殖与呼吸综合征病毒（PRRSV）和寨卡病毒（ZIKV）的最佳复制是必需的，这两种病毒都是在细胞质中复制的正链单链 RNA 病毒。感染病毒的细胞中的 KPNA6 蛋白水平明显高于 mock 感染对照，而 KPNA6 转录物保持稳定。病毒感染阻断了 KPNA6 的泛素-蛋白酶体降解，导致 KPNA6 半衰期延长，细胞中 KPNA6 水平升高。PRRSV nsp12 蛋白诱导 KPNA6 稳定。KPNA6 沉默对 PRRSV 的复制有害，而 KPNA6 敲除则削弱了 ZIKV 的复制。此外，KPNA6 敲除阻断了 PRRSV nsp1β 的核转位，但对具有核定位的另外两种 PRRSV 蛋白的影响最小。在 KPNA6 敲除细胞中外源恢复 KPNA6 表达可恢复 PRRSV nsp1β 的核转位和 ZIKV 的复制。这些结果表明 KPNA6 是 PRRSV 和 ZIKV 复制的重要细胞因子。正链单链 RNA（+ssRNA）病毒在感染细胞的细胞质中复制。运输因子在+ssRNA 病毒核质转运系统中的作用尚不清楚。在这项研究中，我们发现 PRRSV 和 ZIKV 病毒需要运输因子之一的核输入蛋白 α6（KPNA6）来增强病毒复制。我们的数据表明，病毒感染通过病毒干扰 KPNA6 的泛素-蛋白酶体降解来延长 KPNA6 的半衰期，从而导致 KPNA6 蛋白水平升高。值得注意的是，KPNA6 沉默或敲除显著降低了 PRRSV 和 ZIKV 的复制。PRRSV nsp1β 依赖 KPNA6 进入细胞核。此外，在 KPNA6 敲除细胞中外源恢复 KPNA6 表达可导致 nsp1β 核转位和 ZIKV 复制的恢复。这些结果揭示了病毒-细胞相互作用的一个新方面，可能有助于开发新的抗病毒治疗方法。
