Modulation of the Hepatitis C Virus RNA-dependent RNA Polymerase Activity by the Non-Structural (NS) 3 Helicase and the NS4B Membrane Protein

Abstract

The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is believed to be the central catalytic enzyme responsible for HCV replication but there are many unanswered questions about how its activity is controlled. In this study we reveal that two other HCV proteins, NS3 (a protease/helicase) and NS4B (a hydrophobic protein of unknown function), physically and functionally interact with the NS5B polymerase. We describe a new procedure for generating highly pure NS4B, and use this protein in biochemical studies together with NS5B and NS3. To study the functional effects of the protein-protein interactions, we have developed an in vitro replication assay using the natural noncoding 3 regions of the respective positive ((؉)-3 -untranslated region) and negative ((؊)-3 -terminal region) RNA strands of the HCV genome. Our studies show that NS3 dramatically modulates template recognition by NS5B and changes the synthetic products generated by this enzyme. The use of an NTPase-deficient mutant form of NS3 demonstrates that the NTPase activity (and thus helicase activity) of this protein is specifically required for these effects. Moreover, NS4B is found to be a negative regulator of the NS3-NS5B replication complex. Overall, these results reveal that NS3, NS4B, and NS5B can interact to form a regulatory complex that could feature in the process of HCV replication.Hepatitis C virus (HCV) 1 is a major pathogen of parenterally transmitted non-A, non-B hepatitis (1) and often causes the development of malignant chronic disease, including liver cirrhosis and hepatocellular carcinoma (2). With nearly 3% of the population of the world infected with HCV and no protective vaccine available at present, this disease has emerged as a serious global health problem since the virus was first identified (3, 4). HCV is a positive-stranded RNA virus with a genome of ϳ9400 bp. This genomic RNA initially directs the synthesis of at least 10 structural and nonstructural viral proteins (5). Following that, it is utilized by the viral RNA-dependent RNA polymerase (the nonstructural protein 5B; NS5B) as template to generate a complementary negative-stranded RNA. Once synthesized, the negative strands are transcribed into new molecules of positive-stranded genomic RNA, which in turn provide additional templates for viral protein synthesis as well as genomic RNA for the production of progeny virus (5, 6). However, the molecular events that mediate this process remain largely unclear.Several attempts to dissect the mechanistic details of the viral replication cycle have been reported to date. The focal point of such investigations has been NS5B, which possesses an RNA-dependent RNA polymerase (RdRp) activity and is believed to be the key enzyme catalyzing HCV RNA synthesis (7-14). Its crystal structure reveals that it contains the classical finger, palm, and thumb subdomains of the polymerases with the unique feature of a more fully enclosed active site tunnel (15)(16)(17), and a recent report by Bressanelli and colleagues (18) has provided add...

The Nonstructural Protein 3 Protease/Helicase Requires an Intact Protease Domain to Unwind Duplex RNA Efficiently

“…Both full-length NS3 and a truncated helicase fragment have been shown specifically to bind sequences in the HCV genome, and the protease is apparently not required for this interaction (42). In another interesting study that showed an ability of NS3 to stimulate RNA synthesis catalyzed by the HCV NS5B RNA-dependent RNA polymerase, both full-length and truncated NS3 behaved similarly (24). In contrast to these studies, Howe et al (43) reported that a single chain recombinant protein where NS4A is attached to the N terminus of NS3 was more active in helicase assays than either NS3 or a truncated protein.…”

“…Although the HCV helicase has been analyzed both as an isolated fragment and as part of the full-length NS3 protein, the few studies that directly compared the HCV helicase with and without its attached protease have not noted clear differences (11,23,24). There are nevertheless several noteworthy differences between studies that utilize full-length NS3 and those that use recombinant HCV helicase lacking the protease domain.…”

The Serine Protease Domain of Hepatitis C Viral NS3 Activates RNA Helicase Activity by Promoting the Binding of RNA Substrate

“…NS3 helicase activity may be required to assist the viral polymerase during replication of the HCV RNA genome. NS3 may unwind complex RNA structures within the genome, whereas the NS5B viral polymerase copies the RNA (9). There may also be a role for NS3 in viral capsid assembly and the packaging of the RNA genome within (10).…”

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

“…HCV NS3 helicase with an RNAduplex unwinding activity and a binding affinity to NS5B polymerase (Du et al, 2002) has also been shown to interact with HCV X-RNA and 3'-UTR (Kanai et al, 1995;Banerjee and Dasgupta, 2001). Addition of NS3 helicase to the polymerase reaction with the 3'-UTR of HCV genome yielded high molecular weight products without supporting cyclic replication of RNA template (Piccininni et al, 2002). It is still remained to be answered whether the NS3 helicase can unwind the heavily structured X-RNA at the 3'-end of the HCV genome and the double-stranded replication intermediate generated during HCV RNA replication to allow the NS5B to efficiently copy the RNA template in a cyclic manner.…”