Regulation of hepatitis C virion production via phosphorylation of the NS5A protein

Hepatitis C virus (HCV) is a significant pathogen, infecting some 170 million people worldwide. Persistent virus infection often leads to cirrhosis and liver cancer. In the infected cell many RNA directed processes must occur to maintain and spread infection. Viral genomic RNA is constantly replicating, serving as template for translation, and being packaged into new virus particles; processes that cannot occur simultaneously. Little is known about the regulation of these events. The viral NS5A phosphoprotein has been proposed as a regulator of events in the HCV life cycle for years, but the details have remained enigmatic. NS5A is a three-domain protein and the requirement of domains I and II for RNA replication is well documented. NS5A domain III is not required for RNA replication, and the function of this region in the HCV lifecycle is unknown. We have identified a small deletion in domain III that disrupts the production of infectious virus particles without altering the efficiency of HCV RNA replication. This deletion disrupts virus production at an early stage of assembly, as no intracellular virus is generated and no viral RNA and nucleocapsid protein are released from cells. Genetic mapping has indicated a single serine residue within the deletion is responsible for the observed phenotype. This serine residue lies within a casein kinase II consensus motif, and mutations that mimic phosphorylation suggest that phosphorylation at this position regulates the production of infectious virus. We have shown by genetic silencing and chemical inhibition experiments that NS5A requires casein kinase II phosphorylation at this position for virion production. A mutation that mimics phosphorylation at this position is insensitive to these manipulations of casein kinase II activity. These data provide the first evidence for a function of the domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV. The ability to uncouple virus production from RNA replication, as described herein, may be useful in understanding HCV assembly and may be therapeutically important.     

Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly

Persistent infection with the hepatitis C virus (HCV) is a major risk factor for the development of liver cirrhosis and hepatocellular carcinoma. With an estimated about 3% of the world population infected with this virus, the lack of a prophylactic vaccine and a selective therapy, chronic hepatitis C currently is a main indication for liver transplantation. The establishment of cell-based replication and virus production systems has led to first insights into the functions of HCV proteins. However, the role of nonstructural protein 5A (NS5A) in the viral replication cycle is so far not known. NS5A is a membrane-associated RNA-binding protein assumed to be involved in HCV RNA replication. Its numerous interactions with the host cell suggest that NS5A is also an important determinant for pathogenesis and persistence. In this study we show that NS5A is a key factor for the assembly of infectious HCV particles. We specifically identify the C-terminal domain III as the primary determinant in NS5A for particle formation. We show that both core and NS5A colocalize on the surface of lipid droplets, a proposed site for HCV particle assembly. Deletions in domain III of NS5A disrupting this colocalization abrogate infectious particle formation and lead to an enhanced accumulation of core protein on the surface of lipid droplets. Finally, we show that mutations in NS5A causing an assembly defect can be rescued by trans-complementation. These data provide novel insights into the production of infectious HCV and identify NS5A as a major determinant for HCV assembly. Since domain III of NS5A is one of the most variable regions in the HCV genome, the results suggest that viral isolates may differ in their level of virion production and thus in their level of fitness and pathogenesis.     

Interfering with hepatitis C virus assembly in vitro using affinity peptides directed towards core protein

Viral assembly is a crucial key step in the life cycle of every virus. In the case of Hepatitis C virus (HCV), the core protein is the only structural protein to interact directly with the viral genomic RNA. Purified recombinant core protein is able to self-assemble in vitro into nucleocapsid-like particles upon addition of a structured RNA, providing a robust assay with which to study HCV assembly. Inhibition of self-assembly of the C170 core protein (first 170 amino acids) was tested using short peptides derived from the HCV core, from HCV NS5A protein, and from diverse proteins (p21 and p73) known to interact with HCV core protein. Interestingly, peptides derived from the core were the best inhibitors. These peptides are derived from regions of the core predicted to be involved in the interaction between core subunits during viral assembly. We also demonstrated that a peptide derived from the C-terminal end of NS5A protein moderately inhibits the assembly process.     

Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A

Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles.     

Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly

The hepatitis C virus (HCV) is a flavivirus replicating in the cytoplasm of infected cells. The HCV genome is a single-stranded RNA encoding a polyprotein that is cleaved by cellular and viral proteases into 10 different products. While the structural proteins core protein, envelope protein 1 (E1) and E2 build up the virus particle, most nonstructural (NS) proteins are required for RNA replication. One of the least studied proteins is NS2, which is composed of a C-terminal cytosolic protease domain and a highly hydrophobic N-terminal domain. It is assumed that the latter is composed of three trans-membrane segments (TMS) that tightly attach NS2 to intracellular membranes. Taking advantage of a system to study HCV assembly in a hepatoma cell line, in this study we performed a detailed characterization of NS2 with respect to its role for virus particle assembly. In agreement with an earlier report ( Jones, C. T., Murray, C. L., Eastman, D. K., Tassello, J., and Rice, C. M. (2007) J. Virol. 81, 8374-8383 ), we demonstrate that the protease domain, but not its enzymatic activity, is required for infectious virus production. We also show that serine residue 168 in NS2, implicated in the phosphorylation and stability of this protein, is dispensable for virion formation. In addition, we determined the NMR structure of the first TMS of NS2 and show that the N-terminal segment (amino acids 3-11) forms a putative flexible helical element connected to a stable alpha-helix (amino acids 12-21) that includes an absolutely conserved helix side in genotype 1b. By using this structure as well as the amino acid conservation as a guide for a functional study, we determined the contribution of individual amino acid residues in TMS1 for HCV assembly. We identified several residues that are critical for virion formation, most notably a central glycine residue at position 10 of TMS1. Finally, we demonstrate that mutations in NS2 blocking HCV assembly can be rescued by trans-complementation.     

NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.     

Regulation of the production of infectious genotype 1a hepatitis C virus by NS5A domain III

Although hepatitis C virus (HCV) assembly remains incompletely understood, recent studies with the genotype 2a JFH-1 strain suggest that it is dependent upon the phosphorylation of Ser residues near the C terminus of NS5A, a multifunctional nonstructural protein. Since genotype 1 viruses account for most HCV disease yet differ substantially in sequence from that of JFH-1, we studied the role of NS5A in the production of the H77S virus. While less efficient than JFH-1, genotype 1a H77S RNA produces infectious virus when transfected into permissive Huh-7 cells. The exchange of complete NS5A sequences between these viruses was highly detrimental to replication, while exchanges of the C-terminal domain III sequence (46% amino acid sequence identity) were well tolerated, with little effect on RNA synthesis. Surprisingly, the placement of the H77S domain III sequence into JFH-1 resulted in increased virus yields; conversely, H77S yields were reduced by the introduction of domain III from JFH-1. These changes in infectious virus yield correlated well with changes in the abundance of NS5A in RNA-transfected cells but not with RNA replication or core protein expression levels. Alanine replacement mutagenesis of selected Ser and Thr residues in the C-terminal domain III sequence revealed no single residue to be essential for infectious H77S virus production. However, virus production was eliminated by Ala substitutions at multiple residues and could be restored by phosphomimetic Asp substitutions at these sites. Thus, despite low overall sequence homology, the production of infectious virus is regulated similarly in JFH-1 and H77S viruses by a conserved function associated with a C-terminal Ser/Thr cluster in domain III of NS5A.     

Raf-1 kinase associates with Hepatitis C virus NS5A and regulates viral replication

Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infection associated with severe liver disease. HCV nonstructural protein 5A (NS5A) is essential for viral replication. Here, the kinase Raf-1 was identified as a novel cellular binding partner of NS5A, binding to the C-terminal domain of NS5A. Raf-1 colocalizes with NS5A in the HCV replication complex. The interaction of NS5A with Raf-1 results in increased Raf-1 phosphorylation at serine 338. Integrity of Raf-1 is crucial for HCV replication: inhibition of Raf-1 by the small-molecule inhibitor BAY43-9006 or downregulation of Raf-1 by siRNA attenuates viral replication.     

Phosphorylation of serine-rich protein encoded by open reading frame 3 of the TT virus genome

TT virus (TTV) is a newly discovered human virus with a single-stranded, circular DNA genome. The TTV DNA sequence includes two major open reading frames (ORFs), ORF1 and ORF2. Recently, spliced TTV mRNAs were detected and revealed two additional coding regions, ORF3 and ORF4. We found sequence similarity between the TTV ORF3 protein and hepatitis C virus (HCV) nonstructural 5A (NS5A) protein, which is a phosphoprotein and is thought to associate with various cellular proteins. To test whether the TTV ORF3 protein is phosphorylated, the state of phosphorylation was analyzed with a transient protein production system. The TTV ORF3 protein was phosphorylated at the serine residues in its C-terminal portion. Furthermore, the TTV ORF3 gene generated two forms of proteins with a different phosphorylation state, similar to the HCV NS5A region, suggesting that TTV ORF3 protein has function(s) similar to phosphorylated viral proteins such as the HCV NS5A protein.     

Direct binding of a hepatitis C virus inhibitor to the viral capsid protein

Over 130 million people are infected chronically with hepatitis C virus (HCV), which, together with HBV, is the leading cause of liver disease. Novel small molecule inhibitors of Hepatitis C virus (HCV) are needed to complement or replace current treatments based on pegylated interferon and ribavirin, which are only partially successful and plagued with side-effects. Assembly of the virion is initiated by the oligomerization of core, the capsid protein, followed by the interaction with NS5A and other HCV proteins. By screening for inhibitors of core dimerization, we previously discovered peptides and drug-like compounds that disrupt interactions between core and other HCV proteins, NS3 and NS5A, and block HCV production. Here we report that a biotinylated derivative of SL209, a prototype small molecule inhibitor of core dimerization (IC(50) of 2.80 μM) that inhibits HCV production with an EC(50) of 3.20 μM, is capable of penetrating HCV-infected cells and tracking with core. Interaction between the inhibitors, core and other viral proteins was demonstrated by SL209-mediated affinity-isolation of HCV proteins from lysates of infected cells, or of the corresponding recombinant HCV proteins. SL209-like inhibitors of HCV core may form the basis of novel treatments of Hepatitis C in combination with other target-specific HCV drugs such as inhibitors of the NS3 protease, the NS5B polymerase, or the NS5A regulatory protein. More generally, our work supports the hypothesis that inhibitors of viral capsid formation might constitute a new class of potent antiviral agents, as was recently also shown for HIV capsid inhibitors.     

Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain

Nonstructural protein 5A of the hepatitis C virus (HCV) is a highly phosphorylated molecule implicated in multiple interactions with the host cell and most likely involved in RNA replication. Two phosphorylated variants of NS5A have been described, designated according to their apparent molecular masses (in kilodaltons) as p56 and p58, which correspond to the basal and hyperphosphorylated forms, respectively. With the aim of identifying a possible role of NS5A phosphorylation for RNA replication, we performed an extensive mutation analysis of three serine clusters that are involved in phosphorylation and hyperphosphorylation of NS5A. In most cases, alanine substitutions for serine residues in the central cluster 1 that enhanced RNA replication to the highest levels led to a reduction of NS5A hyperphosphorylation. Likewise, several highly adaptive mutations in NS4B, which is also part of the replication complex, resulted in a reduction of NS5A hyperphosphorylation too, arguing that alterations of the NS5A phosphorylation pattern play an important role for RNA replication. On the other hand, a deletion encompassing all highly conserved serine residues in the C-terminal region of NS5A that are involved in basal phosphorylation did not significantly affect RNA replication but reduced formation of p56. This region was found to tolerate even large insertions with only a moderate effect on replication. Based on these results, we propose a model of the role of NS5A phosphorylation in the viral life cycle.     

The C terminus of hepatitis C virus NS4A encodes an electrostatic switch that regulates NS5A hyperphosphorylation and viral replication

Hepatitis C virus (HCV) nonstructural protein 4A (NS4A) is only 54 amino acids (aa) in length, yet it is a key regulator of the essential serine protease and RNA helicase activities of the NS3-4A complex, as well as a determinant of NS5A phosphorylation. Here we examine the structure and function of the C-terminal acidic region of NS4A through site-directed mutagenesis of a Con1 subgenomic replicon and through biophysical characterization of a synthetic peptide corresponding to this region. Our genetic studies revealed that in 8 of the 15 C-terminal residues of NS4A, individual Ala substitutions or charge reversal substitutions led to severe replication phenotypes, as well as decreased NS5A hyperphosphorylation. By selecting for replication-competent mutants, several second-site changes in NS3 were identified and shown to suppress these defects in replication and NS5A hyperphosphorylation. Circular-dichroism spectroscopy and nuclear magnetic resonance spectroscopy on a peptide corresponding to the C-terminal 19 aa of NS4A revealed that this region can adopt an alpha-helical conformation, but that this folding requires neutralization of a cluster of acidic residues. Taken together, these data suggest that the C terminus of NS4A acts as a dynamic regulator of NS3-4A interaction, NS5A hyperphosphorylation, and HCV replicase activity.     

Alanine scanning of the hepatitis C virus core protein reveals numerous residues essential for production of infectious virus

Hepatitis C virus (HCV) is an important human pathogen affecting an estimated 3% of the world's population. Recent advances have enabled in vitro propagation of the virus and allow assembly and egress to be investigated for the first time. As a component of the virion, the HCV core protein likely functions primarily in infectious virus production, although little is known about the determinants of this activity. To investigate the roles of core in the viral life cycle, we performed a comprehensive deletion and alanine scanning mutagenesis study of this protein in the context of a genotype 2a reporter virus. We have confirmed that core protein is essential for infectious virion production and have identified numerous residues required for this role. The infectivity of several assembly-defective core mutants could be rescued by compensatory mutations identified in p7 and NS2, suggesting genetic interactions with core and highlighting the importance of these nonstructural proteins in infectious virion morphogenesis.     

Trafficking of hepatitis C virus core protein during virus particle assembly

Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly.     

Hepatitis C Virus Particle Assembly Involves Phosphorylation of NS5A by the c-Abl Tyrosine Kinase

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is thought to regulate the replication of viral RNA and the assembly of virus particles in a serine/threonine phosphorylation-dependent manner. However, the host kinases that phosphorylate NS5A have not been fully identified. Here, we show that HCV particle assembly involves the phosphorylation of NS5A by the c-Abl tyrosine kinase. Pharmacological inhibition or knockdown of c-Abl reduces the production of infectious HCV (J6/JFH1) particles in Huh-7.5 cells without markedly affecting viral RNA translation and replication. NS5A is tyrosine-phosphorylated in HCV-infected cells, and this phosphorylation is also reduced by the knockdown of c-Abl. Mutational analysis reveals that NS5A tyrosine phosphorylation is dependent, at least in part, on Tyr³³⁰ (Tyr²³⁰⁶ in polyprotein numbering). Mutation of this residue to phenylalanine reduces the production of infectious HCV particles but does not affect the replication of the JFH1 subgenomic replicon. These findings suggest that c-Abl promotes HCV particle assembly by phosphorylating NS5A at Tyr³³⁰.     

Rab18 Binds to Hepatitis C Virus NS5A and Promotes Interaction between Sites of Viral Replication and Lipid Droplets

Hepatitis C virus (HCV) is a single-stranded RNA virus that replicates on endoplasmic reticulum-derived membranes. HCV particle assembly is dependent on the association of core protein with cellular lipid droplets (LDs). However, it remains uncertain whether HCV assembly occurs at the LD membrane itself or at closely associated ER membranes. Furthermore, it is not known how the HCV replication complex and progeny genomes physically associate with the presumed sites of virion assembly at or near LDs. Using an unbiased proteomic strategy, we have found that Rab18 interacts with the HCV nonstructural protein NS5A. Rab18 associates with LDs and is believed to promote physical interaction between LDs and ER membranes. Active (GTP-bound) forms of Rab18 bind more strongly to NS5A than a constitutively GDP-bound mutant. NS5A colocalizes with Rab18-positive LDs in HCV-infected cells, and Rab18 appears to promote the physical association of NS5A and other replicase components with LDs. Modulation of Rab18 affects genome replication and possibly also the production of infectious virions. Our results support a model in which specific interactions between viral and cellular proteins may promote the physical interaction between membranous HCV replication foci and lipid droplets.     

Hepatitis C virus NS2 protein serves as a scaffold for virus assembly by interacting with both structural and nonstructural proteins

Many aspects of the assembly of hepatitis C virus (HCV) remain incompletely understood. To characterize the role of NS2 in the production of infectious virus, we determined NS2 interaction partners among other HCV proteins during productive infection. Pulldown assays showed that NS2 forms complexes with both structural and nonstructural proteins, including E1, E2, p7, NS3, and NS5A. Confocal microscopy also demonstrated that NS2 colocalizes with E1, E2, and NS5A in dot-like structures near lipid droplets. However, NS5A did not coprecipitate with E2 and interacted only weakly with NS3 in pulldown assays. Also, there was no demonstrable interaction between p7 and E2 or NS3 in such assays. Therefore, NS2 is uniquely capable of interacting with both structural and nonstructural proteins. Among mutations in p7, NS2, and NS3 that prevent production of infectious virus, only p7 mutations significantly reduced NS2-mediated protein interactions. These p7 mutations altered the intracellular distribution of NS2 and E2 and appeared to modulate the membrane topology of the C-terminal domain of NS2. These results suggest that NS2 acts to coordinate virus assembly by mediating interactions between envelope proteins and NS3 and NS5A within replication complexes adjacent to lipid droplets, where virus particle assembly is thought to occur. p7 may play an accessory role by regulating NS2 membrane topology, which is important for NS2-mediated protein interactions and therefore NS2 function.     

Phosphorylation of hepatitis C virus-encoded nonstructural protein NS5A.

Two proteins, a 56-kDa protein (p56) and a 58-kDa protein (p58), are produced from the hepatitis C virus (HCV) nonstructural region 5A (NS5A). Recently, we found that both proteins are phosphorylated at serine residues and that p58 is a hyperphosphorylated form of p56. Furthermore, hyper-phosphorylation depends on the production of an intact form of the HCV NS4A protein. To clarify the nature of NS5A phosphorylation, pulse-chase analysis was performed with a transient protein production system in cultured cells. The study indicated that basal and hyperphosphorylation of NS5A occurred after proteolytic production of NS5A was complete. In an attempt to identify the location of the hyperphosphorylation sites in p58, proteins with sequential deletions from the C-terminal region of NS5A and with mutations of possible phosphorylated serine residues to a neutral amino acid, alanine, were constructed. The deleted or mutated proteins were then tested for hyperphosphorylation in the presence of the NS4A product. Here, we report that serine residues 2197, 2201, and/or 2204 are important for hyper-phosphorylation. Important sites for basal phosphorylation were identified in the region from residues 2200 to 2250 and in the C-terminal region of the NS5A product. A subcellular localization study showed that most of the NS5A products were localized in the nuclear periplasmic membrane fraction.     

NS3 helicase domains involved in infectious intracellular hepatitis C virus particle assembly

A mutation within subdomain 1 of the hepatitis C virus (HCV) NS3 helicase (NS3-Q221L) (M. Yi, Y. Ma, J. Yates, and S. M. Lemon, J. Virol. 81:629-638, 2007) rescues a defect in production of infectious virus by an intergenotypic chimeric RNA (HJ3). Although NS3-Gln-221 is highly conserved across HCV genotypes, the Leu-221 substitution had no effect on RNA replication or NS3-associated enzymatic activities. However, while transfection of unmodified HJ3 RNA failed to produce either extracellular or intracellular infectious virus, transfection of HJ3 RNA containing the Q221L substitution (HJ3/QL) resulted in rapid accumulation of intracellular infectious particles with release into extracellular fluids. In the absence of the Q221L mutation, both NS5A and NS3 were recruited to core protein on the surface of lipid droplets, but there was no assembly of core into high-density, rapidly sedimenting particles. Further analysis demonstrated that a Q221N mutation minimally rescued virus production and led to a second-site I399V mutation in subdomain 2 of the helicase. Similarly, I399V alone allowed only low-level virus production and led to selection of an I286V mutation in subdomain 1 of the helicase which fully restored virus production, confirming the involvement of both major helicase subdomains in the assembly process. Thus, multiple mutations in the helicase rescue a defect in an early-intermediate step in virus assembly that follows the recruitment of NS5A to lipid droplets and precedes the formation of dense intracellular viral particles. These data reveal a previously unsuspected role for the NS3 helicase in early virion morphogenesis and provide a new perspective on HCV assembly.     

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.