Hepatitis C Virus Core Protein Interacts with Cellular Putative RNA Helicase

Abstract

The nucleocapsid core protein of hepatitis C virus (HCV) has been shown to trans-act on several viral or cellular promoters. To get insight into the trans-action mechanism of HCV core protein, a yeast two-hybrid cloning system was used for identification of core protein-interacting cellular protein. One such cDNA clone encoding the DEAD box family of putative RNA helicase was obtained. This cellular putative RNA helicase, designated CAP-Rf, exhibits more than 95% amino acid sequence identity to other known RNA helicases including human DBX and DBY, mouse mDEAD3, and PL10, a family of proteins generally involved in translation, splicing, development, or cell growth. In vitro binding or in vivo coimmunoprecipitation studies demonstrated the direct interaction of the full-length/matured form and C-terminally truncated variants of HCV core protein with this targeted protein. Additionally, the protein’s interaction domains were delineated at the N-terminal 40-amino-acid segment of the HCV core protein and the C-terminal tail of CAP-Rf, which encompassed its RNA-binding and ATP hydrolysis domains. Immunoblotting or indirect immunofluorescence analysis revealed that the endogenous CAP-Rf was mainly localized in the nucleus and to a lesser extent in the cytoplasm, and when fused with FLAG tag, it colocalized with the HCV core protein either in the cytoplasm or in the nucleus. Similar to other RNA helicases, this cellular RNA helicase has nucleoside triphosphatase-deoxynucleoside triphosphatase activity, but this activity is inhibited by various forms of homopolynucleotides and enhanced by the HCV core protein. Moreover, transient expression of HCV core protein in human hepatoma HuH-7 cells significantly potentiated the trans-activation effect of FLAG-tagged CAP-Rf or untagged CAP-Rf on the luciferase reporter plasmid activity. All together, our results indicate that CAP-Rf is involved in regulation of gene expression and that HCV core protein promotes thetrans-activation ability of CAP-Rf, likely via the complex formation and the modulation of the ATPase-dATPase activity of CAP-Rf. These findings provide evidence that HCV may have evolved a distinct mechanism in alteration of host cellular gene expression regulation via the interaction of its nucleocapsid core protein and cellular putative RNA helicase known to participate in all aspects of cellular processes involving RNA metabolism. This feature of core protein may impart pleiotropic effects on host cells, which may partially account for its role in HCV pathogenesis.

Diversion of stress granules and P-bodies during viral infection

“…Several lines of evidence suggest HCV interact with PBs or PB proteins. Two reports indicated that the HCV core protein forms complexes with DDX3 and colocalizes in cytoplasmic punctae similar to PBs (Mamiya and Worman, 1999;You et al, 1999). Knockdown of DDX3 results in repression of HCV replication, implying an important role in replication (Ariumi et al, 2007).…”

DExD/H-box RNA helicases as mediators of anti-viral innate immunity and essential host factors for viral replication

“…DDX3 was shown to interact with the Hepatitis C Virus (HCV) Core protein, which forms part of the viral nucleocapsid [138][139][140]. While the functional consequences of this interaction have still not been fully established, it was more recently shown that DDX3 is required for HCV replication [90,91].…”

Cytoplasmic RNA Granules and Viral Infection

“…Key steps of HCV replication and assembly take place around lipid droplets. Although HCV core may not directly interact with DDX3 (5), HCV core protein forms complexes containing DDX3 and colocalizes in lipid droplets (72,132). In addition, several other PB components (DDX6 (Rck/p54), Lsm1, PATL1, Ago2, and Xrn1) are co-opted to HCV core-containing assembly sites at lipid droplets (7, 91) ( Fig.…”