Paul David, Madan Vanesa, Ramirez Omar, Bencun Maja, Stoeck Ina Karen, Jirasko Vlastimil, Bartenschlager Ralf
Department of Infectious Diseases, Molecular Virology, Medical Faculty, Heidelberg University, Heidelberg, Germany
Department of Infectious Diseases, Molecular Virology, Medical Faculty, Heidelberg University, Heidelberg, Germany.
J Virol. 2018 Jan 30;92(4). doi: 10.1128/JVI.01890-17. Print 2018 Feb 15.
Hepatitis C virus (HCV) RNA replication occurs in tight association with remodeled host cell membranes, presenting as cytoplasmic accumulations of single-, double-, and multimembrane vesicles in infected cells. Formation of these so-called replication organelles is mediated by a complex interplay of host cell factors and viral replicase proteins. Of these, nonstructural protein 4B (NS4B), an integral transmembrane protein, appears to play a key role, but little is known about the molecular mechanisms of how this protein contributes to organelle biogenesis. Using forward and reverse genetics, we identified glycine zipper motifs within transmembrane helices 2 and 3 of NS4B that are critically involved in viral RNA replication. Foerster resonance energy transfer analysis revealed the importance of the glycine zippers in NS4B homo- and heterotypic self-interactions. Additionally, ultrastructural analysis using electron microscopy unraveled a prominent role of glycine zipper residues for the subcellular distribution and the morphology of HCV-induced double-membrane vesicles. Notably, loss-of-function NS4B glycine zipper mutants prominently induced single-membrane vesicles with secondary invaginations that might represent an arrested intermediate state in double-membrane vesicle formation. These findings highlight a so-far-unknown role of glycine residues within the membrane integral core domain for NS4B self-interaction and functional as well as structural integrity of HCV replication organelles. Remodeling of the cellular endomembrane system leading to the establishment of replication organelles is a hallmark of positive-strand RNA viruses. In the case of HCV, expression of the nonstructural proteins induces the accumulation of double-membrane vesicles that likely arise from a concerted action of viral and coopted cellular factors. However, the underlying molecular mechanisms are incompletely understood. Here, we identify glycine zipper motifs within HCV NS4B transmembrane segments 2 and 3 that are crucial for the protein's self-interaction. Moreover, glycine residues within NS4B transmembrane helices critically contribute to the biogenesis of functional replication organelles and, thus, efficient viral RNA replication. These results reveal how glycine zipper motifs in NS4B contribute to structural and functional integrity of the HCV replication organelles and, thus, viral RNA replication.
丙型肝炎病毒（HCV）RNA复制与重塑的宿主细胞膜紧密相关，在受感染细胞中表现为单膜、双膜和多膜囊泡的细胞质聚集。这些所谓的复制细胞器的形成是由宿主细胞因子和病毒复制酶蛋白的复杂相互作用介导的。其中，非结构蛋白4B（NS4B）是一种整合跨膜蛋白，似乎起着关键作用，但对于该蛋白如何促进细胞器生物发生的分子机制知之甚少。利用正向和反向遗传学，我们在NS4B的跨膜螺旋2和3中鉴定出甘氨酸拉链基序，它们在病毒RNA复制中起关键作用。Förster共振能量转移分析揭示了甘氨酸拉链在NS4B同型和异型自相互作用中的重要性。此外，使用电子显微镜的超微结构分析揭示了甘氨酸拉链残基在HCV诱导的双膜囊泡的亚细胞分布和形态中起重要作用。值得注意的是，功能丧失的NS4B甘氨酸拉链突变体显著诱导具有二次内陷的单膜囊泡，这可能代表双膜囊泡形成中的停滞中间状态。这些发现突出了膜整合核心结构域内甘氨酸残基在NS4B自相互作用以及HCV复制细胞器的功能和结构完整性方面迄今未知的作用。细胞内膜系统的重塑导致复制细胞器的建立是正链RNA病毒的一个标志。就HCV而言，非结构蛋白的表达诱导双膜囊泡的积累，这可能源于病毒和被选用的细胞因子的协同作用。然而，潜在的分子机制尚未完全了解。在这里，我们在HCV NS4B跨膜片段2和3中鉴定出甘氨酸拉链基序，它们对该蛋白的自相互作用至关重要。此外，NS4B跨膜螺旋内的甘氨酸残基对功能性复制细胞器的生物发生至关重要，从而对有效的病毒RNA复制至关重要。这些结果揭示了NS4B中的甘氨酸拉链基序如何促进HCV复制细胞器的结构和功能完整性，进而促进病毒RNA复制。
