Glycine Zipper Motifs in Hepatitis C Virus Nonstructural Protein 4B Are Required for the Establishment of Viral Replication Organelles

Abstract

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.

RACK1 mediates rewiring of intracellular networks induced by hepatitis C virus infection

“…This compartmentalization of replication machinery allows the enrichment and coordination of cellular and viral factors required for RNA replication, and the evasion from innate host defense systems recognizing non-self RNAs such as 5′-triphosphorylated RNAs and double-stranded RNAs [5, 6]. Two distinct morphotypes of replication organelles have been reported, corresponding to the ‘invaginated vesicles/spherule type’ and the ‘double membrane vesicle (DMV) type’ [7]. DMVs are the major component of the HCV replication organelle [8].…”

“…Further investigation into the NS5A-mediated DMV formation revealed that NS5A domain 1 is required for the DMV formation [8]. Other viral protein(s) such as nonstructural protein 4B (NS4B) augments DMV formation activity of NS5A [7].…”

Hepatitis C Viral Replication Complex

“…The observation that NS5A inhibitors (e.g., daclatasvir) block the HCV RO formation independent of RNA replication demonstrated the essential role of NS5A in the RO formation [ 28 ]. The efficiency of the DMV formation induced by NS5A alone is low but is greatly enhanced when the other nonstructural proteins are also expressed, e.g., NS4B, NS3, or NS5B [ 26 , 29 , 30 ].…”

“…Moreover, NS4B contains a GXXXXGK P-loop for nucleotide triphosphate binding, which may be involved in membrane rearrangements [ 36 ]. In addition, NS4B could form homo-oligomeric complexes, which are required for the RO formation [ 29 , 37 , 38 ].…”

Clinical Morphology of the Liver: Hepatocytes, Endomembrane System

“…Мелковезикулярное полотно представляет своеобразный рецепторный «аэродром» (репликативную «фабрику») в виде ремоделированных мембран на которых происходит репликация HCV. Ремоделированные мембраны представляют собой цитоплазматические накопления одно-, двух-и мультимембранных везикул в инфицированных HCV клетках [22,23,24]. Формирование таких репликационных органелл опосредуется сложным взаимодействием факторов клетки-хозяина и вирусных репликативных белков.…”