Poenisch Marion, Metz Philippe, Blankenburg Hagen, Ruggieri Alessia, Lee Ji-Young, Rupp Daniel, Rebhan Ilka, Diederich Kathrin, Kaderali Lars, Domingues Francisco S, Albrecht Mario, Lohmann Volker, Erfle Holger, Bartenschlager Ralf
Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
Department of Computational Biology and Applied Algorithmics, Max Planck Institute for Informatics, Saarbrücken, Germany; Center for Biomedicine, European Academy Bozen/Bolzano (EURAC), Bolzano, Italy, Affiliated Institute of the University of Lübeck, Lübeck, Germany.
PLoS Pathog. 2015 Jan 8;11(1):e1004573. doi: 10.1371/journal.ppat.1004573. eCollection 2015 Jan.
Hepatitis C virus (HCV) is a major cause of chronic liver disease affecting around 130 million people worldwide. While great progress has been made to define the principle steps of the viral life cycle, detailed knowledge how HCV interacts with its host cells is still limited. To overcome this limitation we conducted a comprehensive whole-virus RNA interference-based screen and identified 40 host dependency and 16 host restriction factors involved in HCV entry/replication or assembly/release. Of these factors, heterogeneous nuclear ribonucleoprotein K (HNRNPK) was found to suppress HCV particle production without affecting viral RNA replication. This suppression of virus production was specific to HCV, independent from assembly competence and genotype, and not found with the related Dengue virus. By using a knock-down rescue approach we identified the domains within HNRNPK required for suppression of HCV particle production. Importantly, HNRNPK was found to interact specifically with HCV RNA and this interaction was impaired by mutations that also reduced the ability to suppress HCV particle production. Finally, we found that in HCV-infected cells, subcellular distribution of HNRNPK was altered; the protein was recruited to sites in close proximity of lipid droplets and colocalized with core protein as well as HCV plus-strand RNA, which was not the case with HNRNPK variants unable to suppress HCV virion formation. These results suggest that HNRNPK might determine efficiency of HCV particle production by limiting the availability of viral RNA for incorporation into virions. This study adds a new function to HNRNPK that acts as central hub in the replication cycle of multiple other viruses.
丙型肝炎病毒（HCV）是慢性肝病的主要病因，全球约有1.3亿人受其影响。虽然在确定病毒生命周期的主要步骤方面已取得了很大进展，但关于HCV如何与其宿主细胞相互作用的详细知识仍然有限。为了克服这一局限性，我们进行了一项基于全病毒RNA干扰的全面筛选，确定了40个宿主依赖性因子和16个宿主限制因子参与HCV的进入/复制或组装/释放。在这些因子中，发现异质性核核糖核蛋白K（HNRNPK）可抑制HCV颗粒的产生，而不影响病毒RNA复制。这种对病毒产生的抑制作用是HCV特有的，与组装能力和基因型无关，在相关的登革病毒中未发现。通过使用敲低拯救方法，我们确定了HNRNPK中抑制HCV颗粒产生所需的结构域。重要的是，发现HNRNPK与HCV RNA特异性相互作用，这种相互作用因突变而受损，这些突变也降低了抑制HCV颗粒产生的能力。最后，我们发现，在HCV感染的细胞中，HNRNPK的亚细胞分布发生了改变；该蛋白被募集到脂滴附近的位点，并与核心蛋白以及HCV正链RNA共定位，而无法抑制HCV病毒体形成的HNRNPK变体则不是这种情况。这些结果表明，HNRNPK可能通过限制病毒RNA掺入病毒体的可用性来决定HCV颗粒产生的效率。这项研究为HNRNPK增添了一项新功能，它在多种其他病毒的复制周期中起着核心枢纽的作用。
