Inhibition of Hepatitis C Virus Replication by IFN-Mediated ISGylation of HCV-NS5A Min-Jung Kim and Joo-Yeon Yoo This information is current as J Immunol 2010; 185:4311-4318; Prepublished online 1 of October 2, 2021. September 2010; doi: 10.4049/jimmunol.1000098 http://www.jimmunol.org/content/185/7/4311 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/09/01/jimmunol.100009 Material 8.DC1 References This article cites 50 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/185/7/4311.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Inhibition of Hepatitis C Virus Replication by IFN-Mediated ISGylation of HCV-NS5A Min-Jung Kim and Joo-Yeon Yoo ISG15 is a ubiquitin-like molecule whose expression is induced by type I IFN (IFN-a/b) or in response to virus or bacterial infection. ISG15 or conjugation of ISG15 to target proteins was reported to play critical roles in the regulation of antiviral responses. IFN restricts replication of hepatitis C virus (HCV). However, molecular mechanism of IFN-a/b that inhibits HCV replication is not clear yet. In the current study, we demonstrated that replication of HCV was inhibited by overexpression of ISG15 and ISG15-conjugation enzymes in the HCV subgenomic replicon cells. Among various nonstructural proteins of HCV, NS5A was identified as the substrate for ISGylation. Furthermore, protein stability of NS5A was decreased by overexpression of ISG15 or ISG15-conjugating enzymes. The inhibitory effect of ISG15 or ISGylation on NS5A was efficiently blocked by sub- stitution of lysine at 379 residue to arginine within the C-terminal region, suggesting that ISGylation directly controls protein stability of NS5A. Finally, the inhibitory effect of IFN-a/b on HCV replication was further enhanced by ISGylation, suggesting Downloaded from ISG15 as a therapeutic tool for combined therapy with IFN against HCV. The Journal of Immunology, 2010, 185: 4311–4318. epatitis C virus (HCV) is an enveloped RNA virus that IFN is often observed in patients with HCV infection (12, 13). In- belongs to the genus Hepacivirus, family Flaviviridae terestingly, mutations in HCV-NS5A were reported to correlate (1). The HCV genome is a single-stranded, 9.6-kb–long with responsiveness to IFN therapy in patients, although its precise H http://www.jimmunol.org/ RNA molecule. It encodes a 3000-aa polyprotein that undergoes mechanism is not conclusive (14). To overcome IFN resistance in posttranslational processing to yield at least 10 functionally dis- patients with HCV infection, the development of chemicals that tinct viral proteins: core, envelope protein 1 (E1), envelope protein inhibit HCV-specific enzymatic activities or antisense oligomers 2 (E2), p7, and nonstructural protein (NS)2, NS3, NS4A, NS4B, that inhibit HCV replication has been undertaken (15–17). NS5A, and NS5B (2–4). Unlike other HCV nonstructural proteins, Pathogen-associated molecular patterns of replicating HCV RNA NS5A does not have enzymatic activity. Instead, HCV-NS5A phys- genomes, such as 59-triphosphate RNA or dsRNA, are recognized ically interacts with various cellular proteins, such as p53, IFN- by the product of RIG-I in the cytoplasm. It triggers the activation induced, dsRNA-activated protein kinase, or a novel transcription of intracellular signaling pathways and transcription factors, such factor SNF2-related CBP activator protein, to elicit modulator roles as NF-kB and IFN regulatory factors (18–22). An outcome of these by guest on October 2, 2021 in cell-cycle regulation, cellular transformation, and antiviral im- events is the production of type I IFN (IFN-a/b) (23, 24). Secreted mune responses (5–7). HCV-NS5A is a membrane-associated pro- IFN-a/b induces the activation of the IFN-stimulated gene factor 3 tein that uses its N-terminal amphipathic helix domain and is usually complex, which translocates to the nucleus, where it binds to the found in the HCV RNA replication complex (8, 9). HCV-NS5A is IFN-stimulated response element regions of target genes to direct indispensable for HCV RNA replication, because HCV genome the expression of IFN-stimulated genes (ISGs). Among the genes replicates in the lipid droplet-associated membrane of the endo- induced by IFN, IFN-induced, dsRNA-activated protein kinase, plasmic reticulum (ER) (10). MxA, and ISG15 function as effector molecules in the host cell Chronic infection of HCV is a leading cause of liver disease, response to viral infection (24). Multiple studies have shown a clear including chronic hepatitis, liver cirrhosis, and hepatocellular car- absence or low level of IFN-a/b in the hepatocytes of patients with cinoma (11). With the absence of effective vaccines to prevent HCV chronic HCV infection. In addition, patients with chronic HCV infection, combined treatment of pegylated IFN-a with ribavirin infection show no significant level of ISG induction upon treatment is the most favorable choice for therapy. However, resistance to with IFN (25). Based on these findings, it has been suggested that HCV viral proteins disturb the innate immune response of the host and that altered hepatic ISG expression is associated with liver Department of Life Sciences, Pohang University of Science and Technology, Pohang, pathology. Republic of Korea ISG15 is a type I IFN-inducible, ubiquitin-like protein. Most Received for publication January 13, 2010. Accepted for publication July 29, 2010. components for ISG15 conjugation (ISGylation), including UbelL This work was supported by Grant A080084 from the Korea Healthcare Technology (E1, ISG15-activating enzyme), UbcH8 (E2, ISG15-conjugating en- R&D Project. zyme), and HERC (E3, ISG15 ligase), are highly responsive to IFN- Address correspondence and reprint requests to Dr. Joo-Yeon Yoo, Department of a/b–induced signal transduction (26, 27). Expression of ISG15 and Life Sciences, Pohang University of Science and Technology, Hyouja-dong 31, Po- the conjugation of ISG15 to target proteins are strongly promoted by hang 790-784, Republic of Korea. E-mail address: [email protected]. IFN-a/b treatment, dsRNA, and viral or bacterial infection (28). The online version of this article contains supplemental material. Production of ISG15 or ISGylation is believed to play an important Abbreviations used in this paper: E1, envelope protein 1; E2, envelope protein 2; ER, endoplasmic reticulum; HCV, hepatitis C virus; ISDR, interferon sensitivity-deter- role in establishing the antiviral state of an infected cell. ISG15- mining region; ISG, IFN-stimulated gene; NS, nonstructural protein; poly-IC, deficient mice are highly susceptible to influenza, herpes, and Sind- polyinosinic-polycytidylic acid; POSTECH, Pohang University of Science and Tech- bis viral infections (29), whereas the protective role of overexpressed nology; WCE, whole-cell extracts; WT, wild-type. ISG15 in Sindbis virus-infected IFN-a/bR-deficient mice has been Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 reported (30). It was also reported that ISGylation of cellular proteins www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000098 4312 INHIBITION OF HCV REPLICATION BY ISGylation can be suppressed by infection with influenza B virus through the RNA isolation and analysis interaction of ISG15 with the NS1B viral protein (31). In the current Total RNAwas extracted using TRIzol reagent (Invitrogen) and subjected to study, we investigated the role of ISGylation on HCV-NS5A and its real-time RT-PCR. The primer sequences used for PCR were as follows: implication for viral replication in subgenomic HCV replicon cells. HCV-NS4, 59-ACAACAGGCAGCGTGGTCATT-39 and 59-TTCCACAT- GTGCTTTGCCCA-39 and actin, 59-TCATGAAGGTGACGTTGACATC- 9 9 9 Materials and Methods CGT-3 and 5 -CCTAGAAGCATTTGCGGTGCACGATG-3 . Cell culture and transfection Luciferase assay Huh-7 and COS7 cells were maintained in DMEM (Invitrogen, Grand Cells carrying the Huh-luc/neo-ET HCV subgenomic replicon construct were Island, NY), supplemented with 10% FBS (HyClone, Logan, UT) and 1% transfected with empty vectors or expression vectors for the ISGylation- penicillin/streptomycin (Invitrogen). Huh-5-15 and Huh-luc/neo-ET HCV conjugation system. Cells were lysed 48 h posttransfection, and luciferase (genotype 1b) subgenomic replicon cells (gift of R. Bartenschlager, Uni- activity was measured using a dual-luciferase assay kit (Promega, Madison, versity of Heidelberg, Heidelberg, Germany) were maintained in G418 (600 WI). To ensure that equal amounts of cell lysates were used for luciferase mg/ml; Calbiochem, San Diego, CA)-containing media. Cells were treated assay, protein quantification