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Dimerization of the hepatitis C virus nonstructural protein 4B depends on the integrity of an aminoterminal basic leucine zipper.

ABSTRACT: The hepatitis C virus (HCV) nonstructural (NS) protein 4B is known for protein-protein interactions with virus and host cell factors. Only little is known about the corresponding protein binding sites and underlying molecular mechanisms. Recently, we have predicted a putative basic leucine zipper (bZIP) motif within the aminoterminal part of NS4B. The aim of this study was to investigate the importance of this NS4B bZIP motif for specific protein-protein interactions. We applied in silico approaches for 3D-structure modeling of NS4B-homodimerization via the bZIP motif and identified crucial amino acid positions by multiple sequence analysis. The selected sites were used for site-directed mutagenesis within the NS4B bZIP motif and subsequent co-immunoprecipitation of wild-type and mutant NS4B molecules. Respective interaction energies were calculated for wild-type and mutant structural models. NS4B-homodimerization with a gradual alleviation of dimer interaction from wild-type towards the mutant-dimers was observed. The putative bZIP motif was confirmed by a co-immunoprecipitation assay and western blot analysis. NS4B-NS4B interaction depends on the integrity of the bZIP hydrophobic core and can be abolished due to changes of crucial residues within NS4B. In conclusion, our data indicate NS4B-homodimerization and that this interaction is facilitated by the aminoterminal part containing a bZIP motif.

SUBMITTER: Welker MW

PROVIDER: S-EPMC2974824 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Dimerization of the hepatitis C virus nonstructural protein 4B depends on the integrity of an aminoterminal basic leucine zipper.

Welker Martin-Walter MW Welsch Christoph C Meyer Aline A Antes Iris I Albrecht Mario M Forestier Nicole N Kronenberger Bernd B Lengauer Thomas T Piiper Albrecht A Zeuzem Stefan S Sarrazin Christoph C

Protein science : a publication of the Protein Society 20100701 7

The hepatitis C virus (HCV) nonstructural (NS) protein 4B is known for protein-protein interactions with virus and host cell factors. Only little is known about the corresponding protein binding sites and underlying molecular mechanisms. Recently, we have predicted a putative basic leucine zipper (bZIP) motif within the aminoterminal part of NS4B. The aim of this study was to investigate the importance of this NS4B bZIP motif for specific protein-protein interactions. We applied in silico approa ...[more]

PMID: 20506268

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Project description: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.IMPORTANCE 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.

Dataset Information

Dimerization of the hepatitis C virus nonstructural protein 4B depends on the integrity of an aminoterminal basic leucine zipper.

Publications

Dimerization of the hepatitis C virus nonstructural protein 4B depends on the integrity of an aminoterminal basic leucine zipper.

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