Peptidyl-prolyl isomerase Pin1 is a cellular factor required for hepatitis C virus propagation

The life cycle of hepatitis C virus (HCV) is highly dependent on cellular factors. Using small interfering RNA (siRNA) library screening, we identified peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) as a host factor involved in HCV propagation. Here we demonstrated that silencing of Pin1 expression resulted in decreases in HCV replication in both HCV replicon cells and cell culture-grown HCV (HCVcc)-infected cells, whereas overexpression of Pin1 increased HCV replication. Pin1 interacted with both the NS5A and NS5B proteins. However, Pin1 expression was increased only by the NS5B protein. Both the protein binding and isomerase activities of Pin1 were required for HCV replication. Juglone, a natural inhibitor of Pin1, inhibited HCV propagation by inhibiting the interplay between the Pin1 and HCV NS5A/NS5B proteins. These data indicate that Pin1 modulates HCV propagation and may contribute to HCV-induced liver pathogenesis.     

Hepatitis C virus NS5A protein interacts with phosphatidylinositol 4-kinase type IIIalpha and regulates viral propagation

Hepatitis C Virus (HCV) nonstructural 5A (NS5A) is a pleiotropic protein involved in viral RNA replication and modulation of the cellular physiology in HCV-infected cells. To elucidate the mechanisms of the HCV life cycle, we identified cellular factors interacting with the NS5A protein in HCV-infected cells. Huh7.5 cells were electroporated with HCV Jc1 RNA. Cellular factors associated with HCV NS5A were identified by immunoprecipitation with Dynabead-conjugated NS5A antibody and LC-MS/MS. Phosphatidylinositol 4-kinase type IIIα (PI4KIIIα) was identified as a binding partner for the NS5A protein. NS5A derived from both genotypes 1b and 2a interacted with PI4KIIIα. NS5A interacted with PI4KIIIα through amino acids 401-600 of PI4KIIIα and domain I of NS5A. Interference of the protein interaction between NS5A and PI4KIIIα decreased HCV propagation. Knockdown of PI4KIIIα significantly reduced HCV replication in Huh7 cells harboring the subgenomic replicon and in Huh7.5 cells infected with cell culture grown virus (HCVcc). Silencing of PI4KIIIα further inhibited HCV release into the tissue culture medium. NS5A may recruit PI4KIIIα to the HCV RNA replication complex. These data suggest that PI4KIIIα is an essential host factor that supports HCV proliferation and therefore PI4KIIIα may be a legitimate target for anti-HCV therapy.     

Hepatitis C Virus Nonstructural 5A Protein Interacts with Telomere Length Regulation Protein: Implications for Telomere Shortening in Patients Infected with HCV

Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for viral propagation. Using protein microarray analysis, we identified 90 cellular proteins as HCV nonstructural 5A (NS5A) interacting partners, and selected telomere length regulation protein (TEN1) for further study. TEN1 forms a heterotrimeric complex with CTC and STN1, which is essential for telomere protection and maintenance. Telomere length decreases in patients with active HCV, chronic liver disease, and hepatocellular carcinoma. However, the molecular mechanism of telomere length shortening in HCV-associated disease is largely unknown. In the present study, protein interactions between NS5A and TEN1 were confirmed by immunoprecipitation assays. Silencing of TEN1 reduced both viral RNA and protein expression levels of HCV, while ectopic expression of the siRNA-resistant TEN1 recovered the viral protein level, suggesting that TEN1 was specifically required for HCV propagation. Importantly, we found that TEN1 is re-localized from the nucleus to the cytoplasm in HCV-infected cells. These data suggest that HCV exploits TEN1 to promote viral propagation and that telomere protection is compromised in HCV-infected cells. Overall, our findings provide mechanistic insight into the telomere shortening in HCV-infected cells.     

Human VAP-B is involved in hepatitis C virus replication through interaction with NS5A and NS5B

The hepatitis C virus (HCV) nonstructural protein (NS) 5A is a phosphoprotein that associates with various cellular proteins and participates in the replication of the HCV genome. Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) is known to be a host factor essential for HCV replication by binding to both NS5A and NS5B. To obtain more information on the NS5A protein in HCV replication, we screened human brain and liver libraries by a yeast two-hybrid system using NS5A as bait and identified VAP-B as an NS5A-binding protein. Immunoprecipitation and mutation analyses revealed that VAP-B binds to both NS5A and NS5B in mammalian cells and forms homo- and heterodimers with VAP-A. VAP-A interacts with VAP-B through the transmembrane domain. NS5A interacts with the coiled-coil domain of VAP-B via 70 residues in the N-terminal and 341 to 344 amino acids in the C-terminal polyproline cluster region. NS5A was colocalized with VAP-B in the endoplasmic reticulum and Golgi apparatus. The specific antibody to VAP-B suppressed HCV RNA replication in a cell-free assay. Overexpression of VAP-B, but not of a mutant lacking its transmembrane domain, enhanced the expression of NS5A and NS5B and the replication of HCV RNA in Huh-7 cells harboring a subgenomic replicon. In the HCV replicon cells, the knockdown of endogenous VAP-B by small interfering RNA decreased expression of NS5B, but not of NS5A. These results suggest that VAP-B, in addition to VAP-A, plays an important role in the replication of the HCV genome.     

Lipid Droplet-Binding Protein TIP47 Regulates Hepatitis C Virus RNA Replication through Interaction with the Viral NS5A Protein

The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV) infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web) and assists viral assembly in the close vicinity of lipid droplets (LDs). To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1–31), a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47) as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon), indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A) in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47—via its interaction with NS5A—serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web.     

Hepatitis C Virus Non-Structural Protein 3 Interacts with Cytosolic 5′(3′)-Deoxyribonucleotidase and Partially Inhibits Its Activity

Infection with hepatitis C virus (HCV) is etiologically involved in liver cirrhosis, hepatocellular carcinoma and B-cell lymphomas. It has been demonstrated previously that HCV non-structural protein 3 (NS3) is involved in cell transformation. In this study, a yeast two-hybrid screening experiment was conducted to identify cellular proteins interacting with HCV NS3 protein. Cytosolic 5′(3′)-deoxyribonucleotidase (cdN, dNT-1) was found to interact with HCV NS3 protein. Binding domains of HCV NS3 and cellular cdN proteins were also determined using the yeast two-hybrid system. Interactions between HCV NS3 and cdN proteins were further demonstrated by co-immunoprecipitation and confocal analysis in cultured cells. The cellular cdN activity was partially repressed by NS3 protein in both the transiently-transfected and the stably-transfected systems. Furthermore, HCV partially repressed the cdN activity while had no effect on its protein expression in the systems of HCV sub-genomic replicons and infectious HCV virions. Deoxyribonucleotidases are present in most mammalian cells and involve in the regulation of intracellular deoxyribonucleotides pools by substrate cycles. Control of DNA precursor concentration is essential for the maintenance of genetic stability. Reduction of cdN activity would result in the imbalance of DNA precursor concentrations. Thus, our results suggested that HCV partially reduced the cdN activity via its NS3 protein and this may in turn cause diseases.     

Physical and functional interaction between hepatitis C virus NS5A protein and ovarian tumor protein deubiquitinase 7B

Hepatitis C virus (HCV) NS5A protein plays crucial roles in viral RNA replication, virus assembly, and viral pathogenesis. Although NS5A has no known enzymatic activity, it modulates various cellular pathways through interaction with cellular proteins. HCV NS5A (and other HCV proteins) are reportedly degraded through the ubiquitin–proteasome pathway; however, the physiological roles of ubiquitylation and deubiquitylation in HCV infection are largely unknown. To elucidate the role of deubiquitylation in HCV infection, an attempt was made to identify a deubiquitinase (DUB) that can interact with NS5A protein. An ovarian tumor protein (OTU), deubiquitinase 7B (OTUD7B), was identified as a novel NS5A‐binding protein. Co‐immunoprecipitation analyses showed that NS5A interacts with OTUD7B in both Huh‐7 and HCV RNA replicon cells. Immunofluorescence staining revealed that HCV NS5A protein colocalizes with OTUD7B in the cytoplasm. Moreover, HCV infection was found to enhance the nuclear localization of OTUD7B. The OTUD7B‐binding domain on NS5A was mapped using a series of NS5A deletion mutants. The present findings suggest that the domain I of NS5A is important and the region from amino acid 121 to 126 of NS5A essential for the interaction. Either V121A or V124A mutation in NS5A disrupts the NS5A‐OTUD7B interaction. The results of this in vivo ubiquitylation assay suggest that HCV NS5A enhances OTUD7B DUB activity. Taken together, these results suggest that HCV NS5A protein interacts with OTUD7B, thereby modulating its DUB activity.     

Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.     

Active RNA Replication of Hepatitis C Virus Downregulates CD81 Expression

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.     

Y-box-binding protein 1 interacts with hepatitis C virus NS3/4A and influences the equilibrium between viral RNA replication and infectious particle production

The hepatitis C virus (HCV) NS3/4A protein has several essential roles in the virus life cycle, most probably through dynamic interactions with host factors. To discover cellular cofactors that are co-opted by HCV for its replication, we elucidated the NS3/4A interactome using mass spectrometry and identified Y-box-binding protein 1 (YB-1) as an interacting partner of NS3/4A protein and HCV genomic RNA. Importantly, silencing YB-1 expression decreased viral RNA replication and severely impaired the propagation of the infectious HCV molecular clone JFH-1. Immunofluorescence studies further revealed a drastic HCV-dependent redistribution of YB-1 to the surface of the lipid droplets, an important organelle for HCV assembly. Core and NS3 protein-dependent polyprotein maturation were shown to be required for YB-1 relocalization. Unexpectedly, YB-1 knockdown cells showed the increased production of viral infectious particles while HCV RNA replication was impaired. Our data support that HCV hijacks YB-1-containing ribonucleoparticles and that YB-1-NS3/4A-HCV RNA complexes regulate the equilibrium between HCV RNA replication and viral particle production.     

LXR-alpha/SREBP-1c通路参与HCV NS5A诱导的肝细胞脂质代谢紊乱

肝细胞脂肪变性是丙型肝炎患者的突出病理特征,但丙肝病毒（HCV）诱导脂肪变性的分子机制尚不清楚.为探究HCV非结构蛋白5A（NS5A）参与诱导脂肪变性的可能分子机制,本研究以HCV NS5A转基因小鼠为研究对象,采集3～16月龄NS5A转基因小鼠和同窝非转基因小鼠的肝组织,进行病理学检测,并用气相色谱 质谱（GC-MS）法分析脂质主要成分胆固醇酯的含量.采用RT-PCR法检测肝细胞中与脂质代谢密切相关基因肝X受体（LXR-alpha）、固醇调节元件结合蛋白（SREBP-1c）、脂肪酸合成酶（FAS）、硬脂酰辅酶A去饱和酶1（SCD1）、过氧化物酶体增殖物受体alpha（PPAR-alpha）的mRNA表达水平.结果表明,与同窝非转基因对照小鼠相比,3～5月龄NS5A转基因小鼠的肝组织没有发生显著的病理性变化,但6～16月龄的NS5A转基因小鼠的肝脏发生了显著的脂肪变性（47.1% vs 130%;P=0.003）.与此相一致,胆固醇酯的含量在NS5A转基因小鼠的肝脏中显著升高（P < 0.01）.RT PCR检测结果表明,与对照小鼠相比,14月龄NS5A转基因小鼠肝组织中与脂质代谢相关的基因LXR.alpha、SREBP.1c、FAS、SCD1的mRNA表达水平显著增高（P < 0.05）,而PPAR alpha的表达则没有显著变化（P > 0.05）. 以上结果提示,NS5A在小鼠肝细胞中可能通过调高LXR.alpha/SREBP.1c信号通路相关基因的表达,进而促进脂质重新合成,诱导脂肪变性.     

DNA immunization with a plasmid carrying the gene of hepatitis C virus protein 5A (NS5A) induces an effective cellular immune response

In spite of extensive research, no effective vaccine against hepatitis C virus (HCV) has been developed so far. DNA immunization is a potent technique of vaccine design strongly promoting the cellular arm of immune response. The genes encoding nonstructural HCV proteins (NS2-NS5B) are promising candidates for vaccine development. NS5A is a protein involved in viral pathogenesis, in the induction of immune response, and probably in viral resistance to interferon treatment. The objective of this study was to construct a DNA vaccine encoding NS5A protein and evaluate its immunogenicity. A plasmid encoding a full-size NS5A protein was produced using the pcDNA3.1 (+) vector for eukaryotic expression system. The expression of the NS5A gene was confirmed by immunoperoxidase staining of the transfected eukaryotic cells with anti- NS5A monoclonal antibodies. Triple immunization of mice with the plasmid vaccine induced a pronounced cellular immune response against a broad spectrum of NS5A epitopes as assessed by T-cell proliferation and secretion of antiviral cytokines IFN-γ and IL-2. In T-cell stimulation in vitro experiments, NS5A-derived antigens were modeled by synthetic peptides, recombinant proteins of various genotypes, and phages carrying exposed NS5A peptides. A novel immunomodulator Immunomax showed high adjuvant activity in DNA immunization. The data obtained indicate that the suggested DNA construct has a strong potential in the development of the gene vaccines against hepatitis C.     

Heat Shock Protein 72 Is Associated with the Hepatitis C Virus Replicase Complex and Enhances Viral RNA Replication

The NS5A protein of the hepatitis C virus (HCV) is an integral component of the viral replicase. It also modulates cellular signaling and perturbs host interferon responses. The multifunctional characteristics of NS5A are mostly attributed to its ability to interact with various cellular proteins. This study aimed to identify the novel cellular factors that interact with NS5A and decipher the significance of this interaction in viral replication. The NS5A-interacting proteins were purified by the tandem affinity purification (TAP) procedure from cells expressing NS5A and identified by mass spectrometry. The chaperone protein Hsp72 was identified herein. In vivo protein-protein interaction was verified by co-immunoprecipitation and an in situ proximity ligation assay. In addition to NS5A, Hsp72 was also associated with other members of the replicase complex, NS3 and NS5B, suggesting that it might be directly involved in the HCV replication complex. Hsp72 plays a positive regulatory role in HCV RNA replication by increasing levels of the replicase complex, which was attributed either to the increased stability of the viral proteins in the replicase complex or to the enhanced translational activity of the internal ribosome entry site of HCV. The fact that the host chaperone protein Hsp72 is involved in HCV RNA replication may represent a therapeutic target for controlling virus production.     

Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication complex on lipid raft

The lipid raft membrane has been shown to be the site of hepatitis C virus (HCV) RNA replication. The mechanism of formation of the replication complex is not clear. We show here that the formation of the HCV RNA replication complex on lipid raft (detergent-resistant membranes) requires interactions among the HCV nonstructural (NS) proteins and may be initiated by the precursor of NS4B, which has the intrinsic property of anchoring to lipid raft membrane. In hepatocyte cell lines containing an HCV RNA replicon, most of the other NS proteins, including NS5A, NS5B, and NS3, were also localized to the detergent-resistant membranes. However, when individually expressed, only NS4B was associated exclusively with lipid raft. In contrast, NS5B and NS3 were localized to detergent-sensitive membrane and cytosolic fractions, respectively. NS5A was localized to both detergent-sensitive and -resistant membrane fractions. Furthermore, we show that a cellular vesicle membrane transport protein named hVAP-33 (the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein), which binds to both NS5A and NS5B, plays a critical role in the formation of HCV replication complex. The hVAP-33 protein is partially associated with the detergent-resistant membrane fraction. The expression of dominant-negative mutants and small interfering RNA of hVAP-33 in HCV replicon cells resulted in the relocation of NS5B from detergent-resistant to detergent-sensitive membranes. Correspondingly, the amounts of both HCV RNA and proteins in the cells were reduced, indicating that hVAP-33 is critical for the formation of HCV replication complex and RNA replication. These results indicate that protein-protein interactions among the various HCV NS proteins and hVAP-33 are important for the formation of HCV replication complex.     

Annexin A2 is involved in the formation of hepatitis C virus replication complex on the lipid raft

The hepatitis C virus (HCV) RNA replicates in hepatic cells by forming a replication complex on the lipid raft (detergent-resistant membrane [DRM]). Replication complex formation requires various viral nonstructural (NS) proteins as well as host cellular proteins. In our previous study (C. K. Lai, K. S. Jeng, K. Machida, and M. M. Lai, J. Virol. 82:8838-8848, 2008), we found that a cellular protein, annexin A2 (Anxa2), interacts with NS3/NS4A. Since NS3/NS4A is a membranous protein and Anxa2 is known as a lipid raft-associated scaffold protein, we postulate that Anxa2 helps in the formation of the HCV replication complex on the lipid raft. Further studies showed that Anxa2 was localized at the HCV-induced membranous web and interacted with NS4B, NS5A, and NS5B and colocalized with them in the perinuclear region. The silencing of Anxa2 decreased the formation of membranous web-like structures and viral RNA replication. Subcellular fractionation and bimolecular fluorescence complementation analysis revealed that Anxa2 was partially associated with HCV at the lipid raft enriched with phosphatidylinositol-4-phosphate (PI4P) and caveolin-2. Further, the overexpression of Anxa2 in HCV-nonsusceptible HEK293 cells caused the enrichment of HCV NS proteins in the DRM fraction and increased the colony-forming ability of the HCV replicon. Since Anxa2 is known to induce the formation of the lipid raft microdomain, we propose that Anxa2 recruits HCV NS proteins and enriches them on the lipid raft to form the HCV replication complex.     

c-Fos regulates hepatitis C virus propagation

Hepatitis C virus (HCV) RNA replication requires cellular factors as well as viral non-structural proteins (NS protein). Using small interfering RNA (siRNA) library screening, we previously identified c-Fos as a host factor involved in HCV propagation. In the present study, we demonstrated that silencing of c-Fos expression resulted in decrease of HCV propagation in cell culture grown HCV (HCVcc)-infected cells; whereas overexpression of c-Fos significantly increased HCV propagation. We further confirmed the positive role of c-Fos in HCV propagation by both HCV-luciferase reporter assay and immunofluorescence analysis. We showed that c-Fos level was upregulated by HCV infection. Furthermore, phorbol 12-myristate 13-acetate (PMA)-induced c-Fos level was synergistically increased by HCV infection. These data suggest that c-Fos acts as a positive regulator of HCV propagation and may contribute to HCV-associated pathogenesis.     

Domain 2 of nonstructural protein 5A (NS5A) of hepatitis C virus is natively unfolded

Nonstructural protein 5A protein (NS5A) of hepatitis C virus (HCV) plays an important role in the regulation of viral replication, interferon resistance, and apoptosis. HCV NS5A comprises three domains. Recently the structure of domain 1 has been determined, revealing a structural scaffold with a novel zinc-binding motif and a disulfide bond. At present, the structures of domains 2 and 3 remain undefined. Domain 2 of HCV NS5A (NS5A-D2) is important for functions of NS5A and involved in molecular interactions with its own NS5B and PKR, a cellular interferon-inducible serine/threonine specific protein kinase. In this study we performed structural analysis of domain 2 by multinuclear nuclear magnetic resonance (NMR) spectroscopy. The analysis of the backbone 1H, 13C, and 15N resonances, 3JHNalpha coupling constants ,and 3D NOE data indicates that NS5A-D2 lacks secondary structural elements and reveals characteristics of unfolded proteins. NMR relaxation parameters confirmed the lack of rigid structure in the domain. The absence of an ordered conformation and the observation of a highly dynamic behavior of NS5A-D2 may provide an underlying molecular basis on its physiological function to allow NS5A-D2 to interact with a variety of biological partners.     

Hepatitis C virus stimulates the phosphatidylinositol 4-kinase III alpha-dependent phosphatidylinositol 4-phosphate production that is essential for its replication

Phosphatidylinositol 4-kinase III alpha (PI4KA) is an essential cofactor of hepatitis C virus (HCV) replication. We initiated this study to determine whether HCV directly engages PI4KA to establish its replication. PI4KA kinase activity was found to be absolutely required for HCV replication using a small interfering RNA transcomplementation assay. Moreover, HCV infection or subgenomic HCV replicons produced a dramatic increase in phosphatidylinositol 4-phosphate (PI4P) accumulation throughout the cytoplasm, which partially colocalized with the endoplasmic reticulum. In contrast, the majority of PI4P accumulated at the Golgi bodies in uninfected cells. The increase in PI4P was not observed after infection with UV-inactivated HCV and did not reflect changes in PI4KA protein or RNA abundance. In an analysis of U2OS cell lines with inducible expression of the HCV polyprotein or individual viral proteins, viral polyprotein expression resulted in enhanced cytoplasmic PI4P production. Increased PI4P accumulation following HCV protein expression was precluded by silencing the expression of PI4KA, but not the related PI4KB. Silencing PI4KA also resulted in aberrant agglomeration of viral replicase proteins, including NS5A, NS5B, and NS3. NS5A alone, but not other viral proteins, stimulated PI4P production in vivo and enhanced PI4KA kinase activity in vitro. Lastly, PI4KA coimmunoprecipitated with NS5A from infected Huh-7.5 cells and from dually transfected 293T cells. In sum, these results suggest that HCV NS5A modulation of PI4KA-dependent PI4P production influences replication complex formation.