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SummaryThe genomic RNA of hepatitis C virus (HCV) encodes the viral polyprotein precursor that undergoes proteolytic cleavage into structural and nonstructural proteins by cellular and the viral NS3 and NS2-3 proteases. Nonstructural protein 4A (NS4A) is a cofactor of the NS3 serine protease and has been demonstrated to inhibit protein synthesis. In this study, GST pull-down assay was performed to examine potential cellular factors that interact with the NS4A protein and are involved in the pathogenesis of HCV. A trypsin digestion followed by LC-MS/MS analysis revealed that one of the GST-NS4A-interacting proteins to be eukaryotic elongation factor 1A (eEF1A). Both the N-terminal domain of NS4A from amino acid residues 1-20, and the central domain from residues 21-34 interacted with eEF1A, but the central domain was the key player involved in the NS4A-mediated translation inhibition. NS4A(21-34) diminished both cap-dependent and HCV IRES-mediated translation in a dose-dependent manner. The translation inhibitory effect of NS4A(21-34) was relieved by the addition of purified recombinant eEF1A in an in vitro translation system. Taken together, NS4A inhibits host and viral translation through interacting with eEF1A, implying a possible mechanism by which NS4A is involved in the pathogenesis and chronic infection of HCV.

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses protease, nucleoside triphosphatase, and helicase activities. Although the enzymatic activities have been extensively studied, the ATP-and RNAbinding domains of the NS3 helicase are not well-characterized. In this study, NS3 proteins with point mutations in the conserved helicase motifs were expressed in Escherichia coli, purified, and analyzed for their effects on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV cross-linking experiments indicate that the lysine residue in the AX 4 GKS motif is directly involved in ATP binding, whereas the NS3 (GR1490DT) mutant in which the arginine-rich motif (1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutation at Arg1488 (R1488A) and was also affected by the K1236E substitution in the AX 4 GKS motif and the R1490A and GR1490DT mutations in the arginine-rich motif. Previously, Arg1490 was suggested, based on the crystal structure of an NS3-deoxyuridine octamer complex, to directly interact with the ␥-phosphate group of ATP. Nevertheless, our functional analysis demonstrated the critical roles of Arg1490 in binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in ATP binding.Hepatitis C virus (HCV) is the major causative agent of posttransfusion and sporadic non-A, non-B hepatitis (7,27). Patients with HCV infection often develop chronic hepatitis that leads to cirrhosis and hepatocellular carcinoma (1, 9, 36). HCV belongs to the family Flaviviridae whose members are enveloped viruses containing a single-stranded positive-sense RNA genome. The HCV genome bears a single open reading frame that encodes a polyprotein encompassing about 3,000 amino acid residues (8, 23, 42). Proteolytic cleavage of the polyprotein by host and viral proteases yields at least 10 products as NH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (15,16,19,21,29,38). HCV nonstructural protein 3 (NS3) is a multifunctional protein that possesses three known enzymatic activities in two independent domains. The N-terminal one-third domain contains a serine protease activity. The NS3 protease and the viral NS2 and NS4A proteins are important for the processing of the HCV polyprotein to generate nonstructural proteins (2,10,14,15,20,43). The C-terminal two-thirds domain of the NS3 protein contains several conserved sequences shared by members of the DEAD box family of RNA helicases (12, 13, 37). These include AX 4 GKS Walker A nucleotide binding motif (motif I), DECH Walker B nucleoside triphosphate (NTP) binding-hydrolysis motif (motif II), TAT RNA unwinding motif (motif III), and a QRRGRTGR arginine-rich motif (motif VI) thought to be involved in RNA binding. NTPase and RNA helicase activities have been demonstrated for both the NS3 helicase domain (24,40,41) and the full-length NS3 protein (11,18). The kinetics of the ATP hydrolysis and duplex unwinding have been analyzed (32-34). In a...

The NS3 protein of hepatitis C virus (HCV) possesses protease activity responsible for the proteolytic cleavage of the viral polyprotein at the junctions of nonstructural proteins downstream of NS3. The NS3 protein was also found to be internally cleaved. In this study, we demonstrated that internal cleavages occurred on the NS3 protein of genotype 1b in the presence of NS4A, both in culture cells and with a mouse model system. No internal cleavage products were detected with the NS3 and NS4A proteins of genotype 2a. Three potential cleavage sites were detected in the NS3 protein (genotype 1b), with IPT 402 ͦS being the major one. The internal cleavage requires the polyprotein processing activity of NS3 protease, but when supplemented in trans, the internal cleavage efficiency is reduced. In addition, several mutations in NS4A disrupted the internal cleavage of NS3 but did not affect polyprotein processing, indicating that NS4A contributes differently to these two proteolytic activities. Furthermore, Ile-25, Val-26, and Ile-29 of the NS4A protein, important for the NS4A-dependent internal cleavages, were also shown to be critical for the transforming activity of NS3, but mutations at these critical residues resulted only in a slight increase of HCV replicating efficiency. The internal cleavage-associated enhancement of the transforming activity of NS3 was reduced when a T402A substitution at the major internal cleavage site was introduced. The multiple roles of NS4A in viral multiplication and pathogenesis make NS4A an ideal molecular target for HCV therapy.Hepatitis C virus (HCV) is a member of the family Flaviviridae with a single-stranded, positive-sense RNA of 9.6 kb that encodes a polyprotein of approximately 3,000 amino acid residues (8,18,35). The polyprotein precursor is further processed into structural and nonstructural proteins by cellular signal peptidase and viral proteases. Cleavage at the NS2-NS3 junction occurs autoproteolytically with the NS2-3 protease (15), whereas cleavage of the downstream nonstructural proteins is mediated by the viral NS3 serine protease (3,10,14).The viral NS3 protein has multiple functions. The Nterminal-one-third region possesses protease activity, and the C-terminal-two-thirds region contains NTPase and RNA helicase activities. In addition, previous studies indicate that the N-terminal domain of the NS3 protein has the potential for cell transformation (30,41,43). Subcutaneous inoculation of nude mice with cells previously transfected with a plasmid that expresses a C-terminal deletion NS3 protein induced tumor formation (16). Mechanisms of NS3 involved in cell transformation are not fully understood. Nevertheless, the transforming ability of NS3 was abolished when cells were treated with the protease inhibitors phenylmethylsulfonyl fluoride and tosylsulfonyl phenylalanyl chloromethyl ketone (43). This indicates that protease activity is required for the transforming activity of the HCV NS3 protein. Furthermore, internal cleavages of the NS3 protein have been reported....

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