A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly

Hughes, Mair; Gretton, Sarah N.; Shelton, Holly; Brown, David D. R.; McCormick, Christopher J.; Angus, Allan G. N.; Patel, Arvind H.; Griffin, Stephen; Harris, Mark

doi:10.1128/jvi.02406-08

Cited by 39 publications

(42 citation statements)

References 33 publications

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“…NS5A contains a highly conserved C-terminal proline motif with the consensus sequence Pro-X-X-Pro-X-Arg that influences both RNA replication and viral assembly (26). This proline motif binds to the SH3 domains of the Src family kinase such as Fyn, Lyn, Hck, and Lck (27).…”

Section: Discussionmentioning

confidence: 99%

Hepatitis C Virus NS5A Protein Interacts with Phosphatidylinositol 4-Kinase Type IIIα and Regulates Viral Propagation

Lim

Hwang

2011

Journal of Biological Chemistry

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Hepatitis C virus (HCV)2 is a single-stranded positive-sense RNA virus capable of establishing a chronic infection in 70 -80% of HCV-infected patients. Approximately 3% of the population of the world are chronically infected with HCV. Over 30% of them will develop cirrhosis within 20 years, which subsequently may lead to hepatocellular carcinoma (1-3). A vaccine is not available and the optimal current therapy for patients infected with HCV is a combination of the pegylated interferon (IFN) and ribavirin. However, the therapy with these agents is prolonged, costly, and associated with a high rate of side effects. Furthermore, the sustained virologic response rate to the combination therapy varies with different genotypes of HCV. Although small molecule inhibitors of viral protease and RNA-dependent RNA polymerase are in clinical development, the error prone nature of the viral RNA polymerase leads to rapid emergence of viral-resistant mutations to these therapeutic candidates. Therefore, a new approach to control HCV proliferation could be identifying the host factors involved in the HCV life cycle. HCV relies heavily on host proteins for all steps of its life cycle, including viral entry, uncoating, replication, assembly, and virion release. Therefore, any step that can interrupt the HCV life cycle will be a putative target for HCV therapy.Nonstructural 5A (NS5A) protein is a multifunctional phosphoprotein consisting of 447 amino acids residues. NS5A protein is localized in the cytoplasm and forms part of the HCV RNA replication complex (4). NS5A has been shown to interact with many host factors, including TRAF2, hVAP-33, PKR, and Grb2, to regulate viral replication and cellular signaling pathways (5-8). A growing body of evidence indicates that NS5A may play important roles in the HCV-induced liver pathogenesis. We have shown that NS5A modulated TNF␣ signaling of the host cells through the NS5A-TRAF2 interaction (5). In addition, we demonstrated that NS5A induced steatosis and hepatocellular carcinoma in transgenic mice (9). Recently, we also showed that NS5A modulated ␤-catenin signaling that might play an important role in HCV pathogenesis (10).Phosphatidylinositol 4-kinase type III␣ (PI4KIII␣) is a lipid kinase that is encoded by the PI4KCA gene in human (11). PI4KIII␣ phosphorylates phosphatidylinositol (PtdIns) to phosphatidylinositol 4-P, which can be further phosphorylated by PIP5 kinases to phosphatidylinositol (4,5)-P 2 . PI4KIII␣ is localized primarily to the endoplasmic reticulum and regulates endoplasmic reticulum exit sites (12, 13). Recently, PI4KIII␣ has been identified as a cellular factor involved in the HCV life cycle using siRNA library screening (14 -19). However, how PI4KIII␣ regulates HCV proliferation is not clearly understood. In this study, we identified PI4KIII␣ as a binding partner for the NS5A protein. Silencing of PI4KIII␣ significantly reduced HCV replication and virion release in HCV-infected cells. These data suggest that HCV may modulate cellular PI4KIII␣ for its own RNA replic...

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Section: Discussionmentioning

confidence: 99%

Hepatitis C Virus NS5A Protein Interacts with Phosphatidylinositol 4-Kinase Type IIIα and Regulates Viral Propagation

Lim

Hwang

2011

Journal of Biological Chemistry

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“…For Western blotting, cells were lysed in GLB as described above at 48 h posttransfection. Virus harvest and titrations were performed as described (41).…”

Section: Methodsmentioning

confidence: 99%

Enhanced hepatitis C virus genome replication and lipid accumulation mediated by inhibition of AMP-activated protein kinase

Mankouri

Tedbury

Gretton

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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133

145

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Hepatitis C virus (HCV) infection is associated with dysregulation of both lipid and glucose metabolism. As well as contributing to viral replication, these perturbations influence the pathogenesis associated with the virus, including steatosis, insulin resistance, and type 2 diabetes. AMP-activated protein kinase (AMPK) plays a key role in regulation of both lipid and glucose metabolism. We show here that, in cells either infected with HCV or harboring an HCV subgenomic replicon, phosphorylation of AMPK at threonine 172 and concomitant AMPK activity are dramatically reduced. We demonstrate that this effect is mediated by activation of the serine/ threonine kinase, protein kinase B, which inhibits AMPK by phosphorylating serine 485. The physiological significance of this inhibition is demonstrated by the observation that pharmacological restoration of AMPK activity not only abrogates the lipid accumulation observed in virus-infected and subgenomic replicon-harboring cells but also efficiently inhibits viral replication. These data demonstrate that inhibition of AMPK is required for HCV replication and that the restoration of AMPK activity may present a target for much needed anti-HCV therapies.

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“…In the earlier study using subgenomic replicon systems, HCV-NS5A was suggested to be involved in HCV RNA replication (8,(35)(36)(37)(38). Because HCV-NS5A contains multiple domains with specific functions, we attempted to determine the responsible domain(s) for regulation by ISG15 conjugation.…”

Section: Hcv-ns5a Is a Target Protein For Isg15 Conjugationmentioning

confidence: 99%

Inhibition of Hepatitis C Virus Replication by IFN-Mediated ISGylation of HCV-NS5A

Kim

Yoo

2010

The Journal of Immunology

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ISG15 is a ubiquitin-like molecule whose expression is induced by type I IFN (IFN-α/β) 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-α/β 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 substitution 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-α/β on HCV replication was further enhanced by ISGylation, suggesting ISG15 as a therapeutic tool for combined therapy with IFN against HCV.

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A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly

Cited by 39 publications

References 33 publications

Hepatitis C Virus NS5A Protein Interacts with Phosphatidylinositol 4-Kinase Type IIIα and Regulates Viral Propagation

Hepatitis C Virus NS5A Protein Interacts with Phosphatidylinositol 4-Kinase Type IIIα and Regulates Viral Propagation

Enhanced hepatitis C virus genome replication and lipid accumulation mediated by inhibition of AMP-activated protein kinase

Inhibition of Hepatitis C Virus Replication by IFN-Mediated ISGylation of HCV-NS5A

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