Class III Phosphatidylinositol 4-Kinase Alpha and Beta Are Novel Host Factor Regulators of Hepatitis C Virus Replication

Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.Hepatitis C virus (HCV) causes liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (52). The HCV genome is a single-stranded RNA molecule where both the 5′ and the 3′ untranslated region (UTR) contain highly conserved RNA structures necessary for polyprotein translation and genome replication (43). The processed polyprotein yields at least three structural proteins and six nonstructural proteins. The structural proteins include the core, which forms the viral nucleocapsid, and the envelope glycoproteins E1 and E2. The viral proteins processed by signal peptidases form viral particles that assemble at the endoplasmic reticulum (ER) and/or Golgi bodies and are released from the host cell by viral budding. The structural protein coding regions are separated from nonstructural proteins by the short membrane peptide p7, thought to function as an ion channel (43, 53). The nonstructural proteins NS2, NS3/4A, NS5A, and NS5B are involved in coordinating the intracellular processes of the virus life cycle, including polyprotein processing and viral RNA replication (34).The Luc-1b cell is a human hepatoma cell line (Huh7) that contains a genotype 1b HCV subgenomic replicon, a luciferase reporter, and a neomycin selection marker, allowing HCV replication to be studied both in vitro and in vivo (8, 36). This subgenomic replicon lacks the coding regions for NS2 and the structural proteins but contains the nonstructural proteins in cis, which are required for replication of the viral RNA. Expression of the luciferase gene acts as a surrogate marker for levels of HCV RNA produced in the cell. The goal of the present study was to use this subgenomic HCV replicon to screen siRNA libraries and identify novel host proteins that are involved in HCV replication.A number of cellular pathways and proteins that play critical roles in HCV replication have recently been described (41, 42, 46). In particular, replication of HCV is tied closely to its localization and transport to various internal membranes and to lipid metabolism (2). Most of the HCV proteins appear to be targeted to the surface of the ER and replication complexes appear to be transported to lipid rafts, where RNA replication can occur (2). Infectious virus particle formation occurs in association with lipid droplets, and this process requires the core and NS5A proteins. In addition, cholesterol pathway production of geranylgeranyl-PP is important to geranylate the FBL2 protein, which serves as a membrane anchor for NS5A (62). The hVAP proteins involved in the localization and trafficking between internal membranous structures are known to be associated with the HCV proteins NS5A and NS5B (59). Thus, host factor lipid metabolism and intracellular protein transport are necessary for HCV replication in cells.Targeting host factors that are required for viral replication offers a strategy to overcome viral resistance and may allow treatment for more than one genotype of HCV and/or a related Flaviviridae virus such as Dengue, West Nile, or yellow fever virus. The current standard-of-care treatment for the genotype 1 strain of HCV infection is pegylated interferon alpha plus ribavirin over a 6-month time course with more than half of infected patients being refractory to this treatment (57). In addition to genotype 1, there are at least five naturally occurring genotype variants of HCV that can complicate a patient''s response to therapy when infected with more than one genotype. As well as the development of mutations, the presence of multiple variants coexisting in patients is thought to contribute to the rapid development of resistance (40). A variety of antiviral therapeutic strategies aim to inhibit viral proteins directly with small molecules or siRNAs (13, 31, 33). Although some small molecule approaches have been successful in preclinical studies, small-molecule strategies directed against the viral targets can still be rendered ineffective due to the development of mutant, treatment-resistant viral strains (13, 40). Thus, combination therapies are a necessary approach to treat the many variants of HCV that exist in the patient population.In the present study, a set of 779 SMARTpool small interfering RNAs (siRNAs) targeting the kinome and 4 siRNAs targeting 5,000 druggable genes (20,000 siRNAs) were tested for their ability to block replication of the Luc-1b HCV subgenomic replicon. siRNAs targeting CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase), a tripartite enzyme that catalyzes the first three steps of pyrimidine biosynthesis, inhibited both the Luc-1b replicon and JFH1-2a virus expression. This activity is consistent with the known inhibitor of this enzyme, leflunomide, which has been shown previously to inhibit both respiratory syncytial virus and HCV (12, 54). siRNAs targeting the mevalonate (diphospho) decarboxylase (MVD) enzyme, which catalyzes the formation of mevalonate, were found to inhibit Luc-1b replication (19). Inhibition of the cholesterol biosynthesis pathway and host cell geranylation has been previously reported to inhibit HCV subgenomic replication (3, 24, 51, 62, 67). siRNA-mediated knockdown of the class III phosphatidylinositol 4-kinases PI4KA and PI4KB inhibited luciferase expression not only for the genotype 1b subgenomic replicons (Luc-1a and Luc-1b) but also for the viral RNA levels of SG-1b, Luc-1b, and Luc-1a. PI4KA knockdown also inhibited Renilla expression in the JFH-1 genotype 2a infectious virus (JFH1-2a), genotype 2a subgenomic replicon (SG-1a), and a genomic and subgenomic genotype 1a replicon (FL-1a and SG-1a). Using the small-molecule inhibitor PIK93 in compound affinity competition experiments and in vitro biochemical assays, we demonstrated PIK93 could bind and inhibit both PI4KA and PI4KB enzymatic activity (58). PIK93 could inhibit luciferase expression in the Luc-1b, Luc-1a, and JFH1-2a infectious virus assays in the submicromolar range. Together, our data suggest that PI4KA and PI4KB regulate HCV replication and that pharmacological inhibition of these enzymes represents a new class of antiviral agents for multiple genotypes of HCV. Finally, since PI4KA and PI4KB are known to regulate protein and lipid transport to and from the ER and Golgi bodies, their function may hold clues as to how movement of HCV replication complexes throughout different organelles is regulated.