The Cellular Antiviral Protein Viperin Is Attenuated by Proteasome-Mediated Protein Degradation in Japanese Encephalitis Virus-Infected Cells

Abstract

Viperin is identified as an antiviral protein induced by interferon (IFN), viral infections, and pathogenassociated molecules. In this study, we found that viperin is highly induced at the RNA level by Japanese encephalitis virus (JEV) and Sindbis virus (SIN) and that viperin protein is degraded in JEV-infected cells through a proteasome-dependent mechanism. Promoter analysis revealed that SIN induces viperin expression in an IFN-dependent manner but that JEV by itself activates the viperin promoter through IFN regulatory factor-3 and AP-1. The overexpression of viperin significantly decreased the production of SIN, but not of JEV, whereas the proteasome inhibitor MG132 sustained the protein level and antiviral effect of viperin in JEVinfected cells. Knockdown of viperin expression by RNA interference also enhanced the replication of SIN, but not that of JEV. Our results suggest that even though viperin gene expression is highly induced by JEV, it is negatively regulated at the protein level to counteract its antiviral effect. In contrast, SIN induces viperin through the action of IFN, and viperin exhibits potent antiviral activity against SIN.Viperin, an interferon (IFN)-induced cellular antiviral protein, was identified originally as cig5 in human cytomegalovirus (HCMV)-infected human fibroblasts (53). A similar gene called vig-1 was identified subsequently in viral hemorrhagic septicemia virus-infected rainbow trout leukocytes (3) and in mouse dendritic cells infected with vesicular stomatitis virus (VSV) and pseudorabies virus (4). Viperin was then found to be induced by both type I and type II IFN and to exhibit antiviral activity against HCMV (9). Furthermore, the results of several gene-profiling microarray studies showed that the viperin gene is one of those that is highly induced by a range of different viruses (20,25,38,40,42,46) and microbial products, such as lipopolysaccharide (35,42), the double-stranded RNA analog poly(I-C) (39), and double-stranded B-form DNA (22), in various cell types.The human viperin gene encodes a protein of 361 amino acids with a predicted molecular mass of 42.2 kDa. Protein sequence analysis reveals a CX 3 CX 2 C motif, which is found in the superfamily of S-adenosyl methionine (SAM)-dependent radical enzymes (44) in residues 83 to 90 of human viperin. Thus, viperin is also called radical SAM domain-containing 2 (RSAD2). The radical SAM superfamily comprises more than 600 members (44, 47), and evidence suggests that the three conserved cysteine residues are part of an unusual iron-sulfur cluster that uses SAM as a cofactor to form a radical that is involved in catalysis (19,27). Although the precise function of viperin remains to be elucidated, recent data have proven its cellular antiviral effects. The overexpression of viperin reduces productive HCMV infection in human fibroblasts by downregulating several structural proteins that are critical for viral assembly and maturation (9), inhibits influenza virus release by perturbing lipid rafts (48), decreases human hepatitis...

The Interferon-Inducible Gene viperin Restricts West Nile Virus Pathogenesis

“…Initial experiments showed that the stable ectopic expression of viperin inhibited HCMV assembly and maturation (7). Subsequent studies showed that the expression of viperin decreased the replication of HCV (20,24), Sindbis virus (6,63), and influenza viruses (60). Analogously, small interfering RNA (siRNA) knockdown of viperin reversed the poly(I ⅐ C)-induced inhibition of HIV replication (41).…”

“…Viperin (RSAD2 or cig5) is an ISG that is expressed in many cell types after exposure to type I and type II IFN, TLR agonists, or virus infection (7,23,41,51,64). Viperin localizes to the cytosolic face of the endoplasmic reticulum (ER) via its amphipathic ␣-helix (21,22), where it is believed to contribute to antiviral effects against hepatitis C virus (HCV) (20,24), Sindbis virus (6,63), HIV (41), influenza virus (60), and human cytomegalovirus (HCMV) (7) by modulating cholesterol and isoprenoid biosynthesis, lipid raft formation, and the composition and localization of lipid droplets (reviewed in reference 16). The ectopic expression of viperin additionally alters ER membrane morphology, which reduces bulk protein secretion and may affect the assembly and secretion of viruses that use an ER-derived exocytic pathway (22).…”

Immunobiology of Japanese Encephalitis Virus

“…Additionally, JEV NS4A protein is reported to block IFN action through inhibiting phosphorylation of Stat 1 and Stat 2 (Lin et al, 2008a). Moreover, aside from IFN antagonism at the level of Jak-Stat signalling, JEV is also able to inhibit a downstream antiviral molecule, viperin, by promoting its degradation via a proteasome-dependent mechanism (Chan et al, 2008).…”

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.…”