Polo-Like Kinase 1 Is Involved in Hepatitis C Virus Replication by Hyperphosphorylating NS5A

Abstract

Hepatitis C virus (HCV) replication involves many viral and host factors. Here, we employed a lentivirusbased RNA interference (RNAi) screening approach to search for possible cellular factors. By using a kinase-phosphatase RNAi library and an HCV replicon reporter system, we identified a serine-threonine kinase, Polo-like kinase 1 (Plk1), as a potential host factor regulating HCV replication. Knockdown of Plk1 reduced both HCV RNA replication and nonstructural (NS) protein production in both HCV replicon cells and HCV-infected cells while it did not significantly affect host cellular growth or cell cycle. Overexpression of Plk1 in the knockdown cells rescued HCV replication. Interestingly, the ratio between the hyperphosphorylated form (p58) and the basal phosphorylated form (p56) of NS5A was lower in the Plk1 knockdown cells and Plk1 kinase inhibitor-treated cells than in the control groups. Further studies showed that Plk1 could be immunoprecipitated together with NS5A. Both proteins partially colocalized in the perinuclear region. Furthermore, Plk1 could phosphorylate NS5A to both the p58 and p56 forms in an in vitro assay system; the phosphorylation efficiency was comparable to that of the reported casein kinase. Taken together, this study shows that Plk1 is an NS5A phosphokinase and thereby indirectly regulates HCV RNA replication. Because of the differential effects of Plk1 on HCV replication and host cell growth, Plk1 could potentially serve as a target for anti-HCV therapy.Hepatitis C virus (HCV) is the major causative agent of non-A/non-B hepatitis (26). More than 170 million people, or 3% of the population in the world, are infected with HCV (29). It establishes chronic infection in at least 85% of infected individuals and is associated with liver cirrhosis and hepatocellular carcinoma. Current treatment, which combines polyethylene glycolinterferon (PEG-IFN) and ribavirin, is ineffective in 22% of patients with non-genotype 1 and in 45% of patients with genotype 1 HCV (1,16,23,55). Therefore, identification of new targets for HCV therapy is an important issue, and cellular genes involved in the HCV life cycle may serve as good candidates.HCV is a positive-strand RNA virus and the only known member of Hepacivirus genus in the family Flaviviridae. Its genome has a length of about 9,600 nucleotides coding for a single polyprotein. The long polyprotein is further processed into at least 10 different products, including four structural proteins (core, E1, E2, and p7) and six nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Nonstructural proteins NS3-NS5B are components of the membrane-associated HCV replication complex (8,13,36,45). NS3 is a bifunctional protein containing an N-terminal protease domain and a C-terminal helicase/NTPase domain, and NS4A serves as a cofactor for NS3 protease. NS4B protein is known to induce intracellular membrane changes that probably serve as the site for viral RNA replication (8). NS5A is required for RNA replication, but little is known about its functi...

Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication

“…CKII was reported to phosphorylate NS5A at Ser-457 at the COOH terminus involved in HCV virion production (30,34). In addition, Polo-like kinase 1 (Plk1) was also reported responsible for NS5A hyperphosphorylation (40). In this study, we showed for the first time that CKI␣ can directly phosphorylate Ser-235 (Fig.…”

Vinexin β Interacts with Hepatitis C Virus NS5A, Modulating Its Hyperphosphorylation To Regulate Viral Propagation

“…By using a hyperphosphorylation inhibitor, at least two host kinases (CK1␣ and Plk1) have been identified that hyperphosphorylate NS5A in vitro and in vivo (59,61). We also found that vinexin ␤ colocalized with Plk1.…”

“…endogenous vinexin ␤ expression inhibited NS5A hyperphosphorylation mediated by CK1␣ in vivo. It has been validated that tight control of NS5A phosphorylation is important for the HCV life cycle, and breaking the balance between the hyperphosphorylated variant (p58) and the hypophosphorylated variant (p56) of NS5A is disastrous for HCV propagation (6,9,15,61). Moreover, CK1␣ has recently been reported to be implicated in NS5A hyperphosphorylation-dependent regulation of HCV virion production (14).…”

“…Thus, investigating the detailed molecular mechanisms by which cellular factors, including some kinases that interact with NS5A and modulate the phosphorylation of NS5A, would be instrumental in the development of antiviral therapy (64,65). Previous studies have identified various host kinases involved in the regulation of the phosphorylation status of NS5A either directly or indirectly, such as CK1␣, CK2␣, Plk1, lipid kinase phosphatidylinositol-4 kinase III alpha, glycogen synthase kinase 3, and MAPK (45,59,61,63,66). Additionally various studies have demonstrated that hyperphosphorylation of NS5A was also modulated by HCV NS proteins (NS3, NS4A, and NS4B) (65,67).…”

“…Therefore, it is commonly believed that any events that disrupt the balance of NS5A phosphorylation are harmful to HCV propagation (6,14,61). For instance, cell culture-adaptive mutations known to decrease the levels of hyperphosphorylated NS5A prevented the efficient infection of a fulllength RNA of HCV in the chimpanzee model (72,73).…”