Abstract
Over the recent years, several homologues with varying degrees of genetic relatedness to hepatitis C virus (HCV) have been identified in a wide range of mammalian species. HCV infectious life cycle relies on a first critical proteolytic event of its single polyprotein, which is carried out by nonstructural protein 2 (NS2) and allows replicase assembly and genome replication. In this study, we characterized and evaluated the conservation of the proteolytic mode of action and regulatory mechanisms of NS2 across HCV and animal hepaciviruses. We first demonstrated that NS2 from equine, bat, rodent, New and Old World primate hepaciviruses also are cysteine proteases. Using tagged viral protein precursors and catalytic triad mutants, NS2 of equine NPHV and simian GBV-B, which are the most closely and distantly related viruses to HCV, respectively, were shown to function, like HCV NS2 as dimeric proteases with two composite active sites. Consistent with the reported essential role for NS3 N-terminal domain (NS3N) as HCV NS2 protease cofactor via NS3N key hydrophobic surface patch, we showed by gain/loss of function mutagenesis studies that some heterologous hepacivirus NS3N may act as cofactors for HCV NS2 provided that HCV-like hydrophobic residues are conserved. Unprecedently, however, we also observed efficient intrinsic proteolytic activity of NS2 protease in the absence of NS3 moiety in the context of C-terminal tag fusions via flexible linkers both in transiently transfected cells for all hepaciviruses studied and in the context of HCV dicistronic full-length genomes. These findings suggest that NS3N acts as a regulatory rather than essential cofactor for hepacivirus NS2 protease. Overall, unique features of NS2 including enzymatic function as dimers with two composite active sites and additional NS3-independent proteolytic activity are conserved across hepaciviruses regardless of their genetic distances, highlighting their functional significance in hepacivirus life cycle.
Highlights
63–79 million individuals were estimated to be chronically infected by hepatitis C virus (HCV) worldwide in 2015 and are at risk of developing severe liver disease including fibrosis, cirrhosis and hepatocellular carcinoma
We demonstrated that nonstructural protein 2 (NS2) from animal hepaciviruses, like HCV NS2, are cysteine proteases, which function as dimers with two composite active sites to ensure a key proteolytic event of the single viral polyprotein at the NS2/ NS3 junction
By using an approach based on the coexpression of proteolytically inactive NS2-NS3 precursors in mammalian cells, we demonstrated that GB virus B (GBV-B) and nonprimate hepacivirus (NPHV) NS2 are dimeric proteases with two composite active sites, highlighting the conservation and the functional significance of this peculiar mode of action among distantly related members of the Hepacivirus genus
Summary
63–79 million individuals were estimated to be chronically infected by hepatitis C virus (HCV) worldwide in 2015 and are at risk of developing severe liver disease including fibrosis, cirrhosis and hepatocellular carcinoma. The only known members of the Hepacivirus genus were HCV and GB virus B (GBV-B), a hepatotropic virus of unknown origin identified in experimentally infected New World primates [2]. A growing number of phylogenetically-related HCV homologues have been identified in the wild in a wide range of mammalian species, including horses [7,8], cattle [9,10], rodents [11,12,13], bats [14] and Old World primates [15]. Pathogenesis and molecular studies are awaited to assess whether these recently identified hepaciviruses have a potential for zoonosis
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