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A locking mechanism regulates RNA synthesis and host protein interaction by the hepatitis C virus polymerase.

ABSTRACT: Mutational analysis of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) template channel identified two residues, Trp(397) and His(428), which are required for de novo initiation but not for extension from a primer. These two residues interact with the Delta1 loop on the surface of the RdRp. A deletion within the Delta1 loop also resulted in comparable activities. The mutant proteins exhibit increased double-stranded RNA binding compared with the wild type, suggesting that the Delta1 loop serves as a flexible locking mechanism to regulate the conformations needed for de novo initiation and for elongative RNA synthesis. A similar locking motif can be found in other viral RdRps. Products associated with the open conformation of the HCV RdRp were inhibited by interaction with the retinoblastoma protein but not cyclophilin A. Different conformations of the HCV RdRp can thus affect RNA synthesis and interaction with cellular proteins.

SUBMITTER: Chinnaswamy S

PROVIDER: S-EPMC2459299 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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A locking mechanism regulates RNA synthesis and host protein interaction by the hepatitis C virus polymerase.

Chinnaswamy Sreedhar S Yarbrough Ian I Palaninathan Satheesh S Kumar C T Ranjith CT Vijayaraghavan Vinodhini V Demeler Borries B Lemon Stanley M SM Sacchettini James C JC Kao C Cheng CC

The Journal of biological chemistry 20080428 29

Mutational analysis of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) template channel identified two residues, Trp(397) and His(428), which are required for de novo initiation but not for extension from a primer. These two residues interact with the Delta1 loop on the surface of the RdRp. A deletion within the Delta1 loop also resulted in comparable activities. The mutant proteins exhibit increased double-stranded RNA binding compared with the wild type, suggesting that the Del ...[more]

PMID: 18442978

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Project description:Host-pathogen interactions are crucial for the successful propagation of pathogens inside the host cell. Knowledge of interactions between host proteins and viral proteins or viral RNA may provide clues for developing novel antiviral strategies. Hepatitis E virus (HEV), a water-borne pathogen that causes acute hepatitis in humans, is responsible for epidemics in developing countries. HEV pathology and molecular biology have been poorly explored due to the lack of efficient culture systems. A contemporary approach, to better understand the viral infection cycle at the molecular level, is the use of system biology tools depicting virus-host interactions. To determine the host proteins which participate in the regulation of HEV replication, we indentified liver cell proteins interacting with HEV RNA at its putative promoter region and those interacting with HEV polymerase (RdRp) protein. We employed affinity chromatography followed by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) to identify the interacting host proteins. Protein-protein interaction networks (PPI) were plotted and analyzed using web-based tools. Topological analysis of the network revealed that the constructed network is potentially significant and relevant for viral replication. Gene ontology and pathway enrichment analysis revealed that HEV RNA promoter- and polymerase-interacting host proteins belong to different cellular pathways such as RNA splicing, RNA metabolism, protein processing in endoplasmic reticulum, unfolded protein response, innate immune pathways, secretory vesicle pathway, and glucose metabolism. We showed that hnRNPK and hnRNPA2B1 interact with both HEV putative promoters and HEV RdRp, which suggest that they may have crucial roles in HEV replication. We demonstrated in vitro binding of hnRNPK and hnRNPA2B1 proteins with the HEV targets in the study, assuring the authenticity of the interactions obtained through mass spectrometry. Thus, our study highlights the ability of viruses, such as HEV, to maneuver host systems to create favorable cellular environments for virus propagation. Studying the host-virus interactions can facilitate the identification of antiviral therapeutic strategies and novel targets.

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A locking mechanism regulates RNA synthesis and host protein interaction by the hepatitis C virus polymerase.

Publications

A locking mechanism regulates RNA synthesis and host protein interaction by the hepatitis C virus polymerase.

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