Identification of Three Interferon-Inducible Cellular Enzymes That Inhibit the Replication of Hepatitis C Virus

Abstract

Hepatitis C virus (HCV) infection is a common cause of chronic hepatitis and is currently treated with alpha interferon (IFN-␣)-based therapies.However, the underlying mechanism of IFN-␣ therapy remains to be elucidated. To identify the cellular proteins that mediate the antiviral effects of IFN-␣, we created a HEK293-based cell culture system to inducibly express individual interferon-stimulated genes (ISGs) and determined their antiviral effects against HCV. By screening 29 ISGs that are induced in Huh7 cells by IFN-␣ and/or up-regulated in HCV-infected livers, we discovered that viperin, ISG20, and double-stranded RNA-dependent protein kinase (PKR) noncytolytically inhibited the replication of HCV replicons. Mechanistically, inhibition of HCV replication by ISG20 and PKR depends on their 3-5 exonuclease and protein kinase activities, respectively. Moreover, our work, for the first time, provides strong evidence suggesting that viperin is a putative radical S-adenosyl-L-methionine (SAM) enzyme. In addition to demonstrating that the antiviral activity of viperin depends on its radical SAM domain, which contains conserved motifs to coordinate [4Fe-4S] cluster and cofactor SAM and is essential for its enzymatic activity, mutagenesis studies also revealed that viperin requires an aromatic amino acid residue at its C terminus for proper antiviral function. Furthermore, although the N-terminal 70 amino acid residues of viperin are not absolutely required, deletion of this region significantly compromises its antiviral activity against HCV. Our findings suggest that viperin represents a novel antiviral pathway that works together with other antiviral proteins, such as ISG20 and PKR, to mediate the IFN response against HCV infection. Hepatitis C virus (HCV) is the sole member of the genusHepacivirus in the family Flaviviridae (43). It establishes persistent infections in the vast majority of infected individuals and is the only known positive-stranded RNA virus that causes persistent life-long infections in humans. Currently, HCV chronically infects more than 170 million people worldwide. Although the initial infection is largely asymptomatic, prolonged infection carries a high risk of chronic hepatitis, cirrhosis, and primary hepatocellular carcinoma (2).Although it has been elegantly demonstrated that HCV can evade the host cellular innate defense response through proteolytic cleavage of RIG-I/MDA5 adaptor protein MAVS and Tolllike receptor 3 adaptor protein TRIF (7,22,25,42,44,48,69), microarray studies performed with liver samples obtained from HCV transiently infected chimpanzees and chronically infected humans revealed that the induction of interferon (IFN)-stimulated genes (ISGs) in HCV-infected livers is a hallmark of the virus infection (5,6,33,39,58,61). These discoveries suggest that the HCV-infected liver is a constant battlefield between the virus and host innate immunity defense systems, and thus IFN-mediated innate responses induced by HCV may play an important role in shaping the pathogenesis and clini...

The antiviral protein, viperin, localizes to lipid droplets via its N-terminal amphipathic α-helix

“…Finally, because viperin was already shown to localize to the cytoplasmic face of the ER by its N-terminal amphipathic ␣-helix, the localization of viperin to lipid droplets provides further information on how lipid droplets may be generated. Viperin has been previously shown to be highly induced upon HCV infection and to limit HCV replication by an unknown mechanism (10,11). In fact, when viperin was overexpressed in HCV replicon-containing cell lines, these cells showed a significant reduction in HCV RNA levels, which were comparable to the HCV RNA levels when the cells were treated with 100 U/mL IFN␣ (11), suggesting that viperin may be one of the most important IFN-induced genes that limits HCV replication.…”

“…Given that several pathogens, notably HCV, are thought to induce and replicate on this intracellular organelle (1), these findings raise possibilities about how the cell responds and fights these infections. Specifically, because viperin has been shown to be induced by HCV infection and to inhibit the replication of this virus (10,11), the localization of viperin to lipid droplets may reflect the mechanism that viperin uses to inhibit HCV. In addition, because the N-terminal amphipathic ␣-helices of both viperin and NS5A are sufficient to target both viperin and dsRed to lipid droplets, it is clear that these helices are lipid droplet-targeting domains and that viperin and NS5A may use the same mechanism of localization.…”

“…Like the HCV NS proteins, viperin has been shown to localize to the cytosolic face of the ER through an N-terminal amphipathic ␣-helix (7,9,12,13). This N-terminal amphipathic ␣-helix is essential for viperin to inhibit HCV and influenza, as mutants lacking this domain have greatly reduced antiviral activity (11,14). Although the precise mechanism by which viperin inhibits HCV is still unknown, viperin was previously shown to inhibit influenza virus budding by disrupting plasma membrane lipid raft microdomains, which are sites of influenza virion assembly and budding (14).…”

“…Viperin is an IFN-induced antiviral protein that is induced upon HCV infection and inhibits HCV replication (10,11). Like the HCV NS proteins, viperin has been shown to localize to the cytosolic face of the ER through an N-terminal amphipathic ␣-helix (7,9,12,13).…”

Fish viperin exerts a conserved antiviral function through RLR-triggered IFN signaling pathway

“…In mammals, viperin was characterized as one of IFN-inducible antiviral effectors (Carlton-Smith and Elliott, 2012;Fitzgerald, 2011;Helbig et al, 2011;Jiang et al, 2008;Mattijssen and Pruijn, 2012;Overby et al, 2009;Quraishi et al, 2010), and its expression regulation has been investigated in mammals (Boudinot et al, 2000;Chan et al, 2008;Chin and Cresswell, 2001;Olofsson et al, 2005;Saitoh et al, 2011;Severa et al, 2006;Stirnweiss et al, 2010). However, this information in fish is still unclear.…”