Differential Requirements of NS4A for Internal NS3 Cleavage and Polyprotein Processing of Hepatitis C Virus

Abstract

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....

Autocatalytic Cleavage within Classical Swine Fever Virus NS3 Leads to a Functional Separation of Protease and Helicase

“…In either case, internal cleavages occur within the helicase domain of NS3 and yield C-terminally truncated molecules. While a single study suggested that cellular proteases mediate the internal NS3 cleavage in HCV (36), it was demonstrated that an autocatalytic protease activity of NS2B/ NS3 or NS3/NS4A is responsible for internal NS3 processing (9,13,37,38).…”

“…As such, proteolytically active single-chain NS4A3 constructs of HCV have been engineered for bacterial expression, representing N-terminal fusions of a central segment of NS4A with NS3 via a short linker sequence (11,12). For HCV, an intramolecular processing of NS3 has been reported that occurs within the helicase domain (13).…”

In trans interaction of hepatitis C virus helicase domains mediates protease activity critical for internal NS3 cleavage and cell transformation

“…Our earlier finding that the internal NS3 cleavage can occur in trans suggested the existence of intermolecular interactions of NS3 proteins [1]. Previous structural analysis also suggested the formation of NS3 helicase dimer [7].…”

Identification of Amino Acid Variants in the Hepatitis C Virus Non-Structural Protein 4A

“…Since the NS4A I25 residue is important, as part of the NS4A central region for enhancing the NS3 protease activity, this residue has been related in experimental models with autoprocessing of NS3 (Butkiewicz et al 1996;Kou et al 2007;Hou et al 2009). In this report, we determined two NS4A variants with I25C substitution from blood donors infected with HCV; the analysis of these NS4A variants suggests that a mutation in I25 affects the protease activity of NS3.…”

“…This linker makes NS4A able to interact with NS3 through a beta-turn as in the natural full-length NS3 (1-631) -NS4A complex. The single-chain protein (NS4A (21-32) -GSGS-NS3 (3-181) ) expressed in Escherichia coli shows enzymatic parameters equivalent to those of the wild type NS3 (1-631) -NS4A protein generated in eukaryotic cells and can be over expressed to high levels as a soluble protein (Dimasi et al 1998;Taremi et al 1998;Howe et al 1999;Leyssen et al 2000;Wang et al 2004;Kou et al 2007). In this study we identified, by DNA sequencing of the 4870-5902 nucleotide region, NS4A amino acid variants from serum samples of HCV infected patients; moreover, we performed amino acid modifications on the 21-32 NS4A sequence in order to understand better the role of NS4 as regulator in HCV replication.…”