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...

For Hepatitis C virus (HCV), initiation of translation is cap-independently mediated by its internal ribosome entry site (IRES). Unlike other IRES-containing viruses that shut off host cap-dependent translation, translation of HCV coexists with that of the host. How HCV IRES-mediated translation is regulated in the infected cells remains unclear. Here, we show that the intracellular level of 40S ribosomal subunit plays a key role in facilitating HCV translation over host translation. In a loss-of-function screen, we identified small subunit ribosomal protein 6 (RPS6) as an indispensable host factor for HCV propagation. Knockdown of RPS6 selectively repressed HCV IRES-mediated translation, but not general translation. Such preferential suppression of HCV translation correlated well with the reduction of the abundance of 40S ribosomal subunit following knockdown of RPS6 or other RPS genes. In contrast, reduction of the amount of ribosomal proteins of the 60S subunit did not produce similar effects. Among the components of general translation machineries, only knockdowns of RPS genes caused inhibitory effects on HCV translation, pointing out the unique role of 40S subunit abundance in HCV translation. This work demonstrates an unconventional notion that the translation initiation of HCV and host possess different susceptibility toward reduction of 40S ribosomal subunit, and provides a model of selective modulation of IRES-mediated translation through manipulating the level of 40S subunit.

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The findings of this study can be used to inform future intervention and guidelines aimed at addressing these barriers systematically and improving adherence to critical care nutrition guidelines for the provision of enteral nutrition.

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Hepatitis C virus (HCV) reorganizes intracellular membranes to establish sites of replication. How viral and cellular proteins target, bind, and rearrange specific membranes into the replication factory remains a mystery. We used a lentivirus-based RNA interference (RNAi) screening approach to identify the potential cellular factors that are involved in HCV replication. A protein with membrane-deforming activity, proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2), was identified as a potential factor. Knockdown of PSTPIP2 in HCV subgenomic replicon-harboring and HCV-infected cells was associated with the reduction of HCV protein and RNA expression. PSTPIP2 was localized predominantly in detergent-resistant membranes (DRMs), which contain the RNA replication complex. PSTPIP2 knockdown caused a significant reduction of the formation of HCV-and NS4B-induced membranous webs. A PSTPIP2 mutant defective in inducing membrane curvature failed to support HCV replication, confirming that the membrane-deforming ability of PSTPIP2 is essential for HCV replication. Taking these results together, we suggest that PSTPIP2 facilitates membrane alterations and is a key player in the formation of the membranous web, which is the site of the HCV replication complex. Hepatitis C virus (HCV), like other RNA viruses, can reorganize cellular membranes to form double-or multimembrane vesicles, including autophagosomes (28) and membranous webs (6). Viral nonstructural proteins (NS3-NS5B), which build up RNA replication complexes (9,22,26), and viral RNA are both associated with membranous webs (6, 9). Membranous webs are accumulations of heterogeneous vesicles derived mainly from the endoplasmic reticulum (ER) membrane (6, 22). These membrane structures are induced by viral proteins and presumably protect the HCV replication complex (RC) from the attack of host nucleases and proteases (20,22). Among all HCV viral proteins, NS4B, which is modified by lipids and has polymerization activity (34), is required for membranous web formation (1,6,17). However, what cellular factors coordinate with NS4B to induce the formation of membranous webs is still unknown.The Pombe Cdc15 homology (PCH) family proteins, such as CIP4 (14) and FCHo (12), are a group of proteins which regulate cytoskeletal and membrane dynamics. They can deform membranes into membrane curvatures during the initiation stage of vesicle formation (27). The membrane-deforming activity is mainly attributed to the intrinsic banana-shaped F-BAR-domain homodimer, which binds to the membrane with its concave surface (8,24). Recent studies also revealed that proteins of the PCH family can interact with lipids, in particular, phosphatidylinositol (PI) (30); for example, FBP17, CIP4, Toca-1, and PSTPIP2 can interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] (31). FBP17 also has binding affinity to phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] (31), and CIP4 can interact with PI3P (14).PSTPIP2 is a 37-kDa ...

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