Component Of Replication Complex Research Articles

LINE-1 expression in cancer correlates with p53 mutation, copy number alteration, and S phase checkpoint

Retrotransposons are genomic DNA sequences that copy themselves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expression. Induction of LINE-1 expression in an invitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway.

Predicting commercially available antiviral drugs that may act on the novel coronavirus (SARS-CoV-2) through a drug-target interaction deep learning model

The infection of a novel coronavirus found in Wuhan of China (SARS-CoV-2) is rapidly spreading, and the incidence rate is increasing worldwide. Due to the lack of effective treatment options for SARS-CoV-2, various strategies are being tested in China, including drug repurposing. In this study, we used our pre-trained deep learning-based drug-target interaction model called Molecule Transformer-Drug Target Interaction (MT-DTI) to identify commercially available drugs that could act on viral proteins of SARS-CoV-2. The result showed that atazanavir, an antiretroviral medication used to treat and prevent the human immunodeficiency virus (HIV), is the best chemical compound, showing an inhibitory potency with Kd of 94.94 nM against the SARS-CoV-2 3C-like proteinase, followed by remdesivir (113.13 nM), efavirenz (199.17 nM), ritonavir (204.05 nM), and dolutegravir (336.91 nM). Interestingly, lopinavir, ritonavir, and darunavir are all designed to target viral proteinases. However, in our prediction, they may also bind to the replication complex components of SARS-CoV-2 with an inhibitory potency with Kd < 1000 nM. In addition, we also found that several antiviral agents, such as Kaletra (lopinavir/ritonavir), could be used for the treatment of SARS-CoV-2. Overall, we suggest that the list of antiviral drugs identified by the MT-DTI model should be considered, when establishing effective treatment strategies for SARS-CoV-2.

Polyprotein processing and intermolecular interactions within the viral replication complex spatially and temporally control norovirus protease activity

Norovirus infections are a major cause of acute viral gastroenteritis and a significant burden on global human health. A vital process for norovirus replication is the processing of the nonstructural polyprotein by a viral protease into the viral components required to form the viral replication complex. This cleavage occurs at different rates, resulting in the accumulation of stable precursor forms. Here, we characterized how precursor forms of the norovirus protease accumulate during infection. Using stable forms of the protease precursors, we demonstrated that all of them are proteolytically active in vitro, but that when expressed in cells, their activities are determined by both substrate and protease localization. Although all precursors could cleave a replication complex-associated substrate, only a subset of precursors lacking the NS4 protein were capable of efficiently cleaving a cytoplasmic substrate. By mapping the full range of protein–protein interactions among murine and human norovirus proteins with the LUMIER assay, we uncovered conserved interactions between replication complex members that modify the localization of a protease precursor subset. Finally, we demonstrate that fusion to the membrane-bound replication complex components permits efficient cleavage of a fused substrate when active polyprotein-derived protease is provided in trans. These findings offer a model for how norovirus can regulate the timing of substrate cleavage throughout the replication cycle. Because the norovirus protease represents a key target in antiviral therapies, an improved understanding of its function and regulation, as well as identification of interactions among the other nonstructural proteins, offers new avenues for antiviral drug design.

Characterization of virus-specific vesicles assembled by West Nile virus non-structural proteins

Flavivirus genome encodes seven non-structural proteins (NSPs) and these NSPs are believed to be involved in their genomic RNA replication, of which the mechanism is unclear. We find that West Nile virus (WNV) NSPs were capable of self-assembling membranous vesicles in cells, which are composed of the host endoplasmic reticulum membrane integrated with viral NS1 and NS4A, and possibly NS2A. The vesicles can further organize into replication complex (RC)-associated vesicles which combine both the vesicle and predicted RC components. The authentic RC-associated vesicles were observed in cells transfected with infectious WNV cDNA as well as WNV replicon. Further mutational analysis showed that WNV/DENV heterologous NS polyproteins derived from lethal chimeric recombinants produced abnormal vesicles. Site-directed mutation of either NS2A or NS4A, which resulted in failure of viral RNA replication, caused immature vesicles too. These findings reveal molecular composition and assembly of the virus-specific nanomachine and confirm that these structures are used for the viral RNA replication.

Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation

Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using ‘APEX2’-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using a customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα.

ID: 126: Interferon-inducible ER protein SCOTIN interferes with hepatitis c virus replication through NS5A protein degradation

An antiviral cytokine interferon- β is induced upon recognition of pathogen-associated molecular patterns expressed by viruses including hepatitis C virus (HCV). Interferon- β induces expression of interferon-stimulated genes which products have cell or viral-regulatory roles. We found a novel interferon- β inducible gene SCOTIN which encodes an endoplasmic reticulum (ER) localized transmembrane protein. Overexpression of SCOTIN inhibited HCV replication in HCV subgenomic replicon cells or HCVcc infected Huh-7 cells. Conversely, knockdown of SCOTIN enhanced the replication of HCV genome. Among HCV replication complex components, HCV NS5A protein which is crucial to maintain HCV genome replication was specifically downregulated by SCOTIN overexpression. Physical interaction between SCOTIN and NS5A was assessed to identify whether SCOTIN affects NS5A protein degradation directly. Transmembrane/proline-rich domain (TMPRD) of SCOTIN which is expected to face cytosol was required for both interaction and degradation of NS5A protein which is anchored to cytoplasmic ER-membrane. Taken together, our results suggest that the interferon- β inducible SCOTIN associates with NS5A protein and promotes NS5A degradation, which helps to limit HCV replication.

Mapping the Interactions between the NS4B and NS3 proteins of dengue virus.

Flavivirus RNA synthesis is mediated by a multiprotein complex associated with the endoplasmic reticulum membrane, named the replication complex (RC). Within the flavivirus RC, NS4B, an integral membrane protein with a role in virulence and regulation of the innate immune response, binds to the NS3 protease-helicase. NS4B modulates the RNA helicase activity of NS3, but the molecular details of their interaction remain elusive. Here, we used dengue virus (DENV) to map the determinants for the NS3-NS4B interaction. Coimmunoprecipitation and an in situ proximity ligation assay confirmed that NS3 colocalizes with NS4B in both DENV-infected cells and cells coexpressing both proteins. Surface plasmon resonance demonstrated that subdomains 2 and 3 of the NS3 helicase region and the cytoplasmic loop of NS4B are required for binding. Using nuclear magnetic resonance (NMR), we found that the isolated cytoplasmic loop of NS4B is flexible, with a tendency to form a three-turn α-helix and two short β-strands. Upon binding to the NS3 helicase, 12 amino acids within the cytoplasmic loop of NS4B exhibited line broadening, suggesting a participation in the interaction. Sequence alignment showed that 4 of these 12 residues are strictly conserved across different flaviviruses. Mutagenesis analysis showed that three (Q134, G140, and N144) of the four evolutionarily conserved NS4B residues are essential for DENV replication. The mapping of the NS3/NS4B-interacting regions described here can assist the design of inhibitors that disrupt their interface for antiviral therapy. NS3 and NS4B are essential components of the flavivirus RC. Using DENV as a model, we mapped the interaction between the viral NS3 and NS4B proteins. The subdomains 2 and 3 of NS3 helicase as well as the cytoplasmic loop of NS4B are critical for the interaction. Functional analysis delineated residues within the NS4B cytoplasmic loop that are crucial for DENV replication. Our findings reveal molecular details of how flavivirus NS3 protein cooperates with NS4B within the RC. In addition, this study has established the rationale and assays to search for inhibitors disrupting the NS3-NS4B interaction for antiviral drug discovery.

Understanding Structural and Dynamic Effects Induced by Key Components of the HCV Polymerase Replication Complex

Hepatitis C virus (HCV) is a wide spread health concern for which there is no vaccine available. HCV contains a single-stranded RNA genome and replicates with the aid of an RNA-dependent RNA polymerase known as non-structural protein 5B (NS5B). NS5B adopts at least two different conformations to replicate viral RNA. The closed conformation is thought to be necessary for initiation of replication while the open conformation is required for elongation of the newly synthesized RNA strand. Transitions between these two conformations play a crucial role in NS5B function. Our goal is to understand how the distribution of conformations sampled by the enzyme is altered during different stages of replication. In previous studies we observed that the free enzyme is able to occupy conformational states that are expected to be relevant for the different stages of RNA replication. Our current studies examine the impact of specific components of the replication complex in shifting the ratio of conformational states adopted by the enzyme. To accomplish this goal we performed Molecular Dynamics simulations of the enzyme bound to various combinations of nucleotides and RNA template. We anticipate that the binding of these components will change the relative population of the different conformations in a way that facilitates enzyme function.

Regulated Transport into the Nucleus of Herpesviridae DNA Replication Core Proteins

The Herpesvirdae family comprises several major human pathogens belonging to three distinct subfamilies. Their double stranded DNA genome is replicated in the nuclei of infected cells by a number of host and viral products. Among the latter the viral replication complex, whose activity is strictly required for viral replication, is composed of six different polypeptides, including a two-subunit DNA polymerase holoenzyme, a trimeric primase/helicase complex and a single stranded DNA binding protein. The study of herpesviral DNA replication machinery is extremely important, both because it provides an excellent model to understand processes related to eukaryotic DNA replication and it has important implications for the development of highly needed antiviral agents. Even though all known herpesviruses utilize very similar mechanisms for amplification of their genomes, the nuclear import of the replication complex components appears to be a heterogeneous and highly regulated process to ensure the correct spatiotemporal localization of each protein. The nuclear transport process of these enzymes is controlled by three mechanisms, typifying the main processes through which protein nuclear import is generally regulated in eukaryotic cells. These include cargo post-translational modification-based recognition by the intracellular transporters, piggy-back events allowing coordinated nuclear import of multimeric holoenzymes, and chaperone-assisted nuclear import of specific subunits. In this review we summarize these mechanisms and discuss potential implications for the development of antiviral compounds aimed at inhibiting the Herpesvirus life cycle by targeting nuclear import of the Herpesvirus DNA replicating enzymes.

The CMG (CDC45/RecJ, MCM, GINS) complex is a conserved component of the DNA replication system in all archaea and eukaryotes

BackgroundIn eukaryotes, the CMG (CDC45, MCM, GINS) complex containing the replicative helicase MCM is a key player in DNA replication. Archaeal homologs of the eukaryotic MCM and GINS proteins have been identified but until recently no homolog of the CDC45 protein was known. Two recent developments, namely the discovery of archaeal GINS-associated nuclease (GAN) that belongs to the RecJ family of the DHH hydrolase superfamily and the demonstration of homology between the DHH domains of CDC45 and RecJ, show that at least some Archaea possess a full complement of homologs of the CMG complex subunits. Here we present the results of in-depth phylogenomic analysis of RecJ homologs in archaea.ResultsWe confirm and extend the recent hypothesis that CDC45 is the eukaryotic ortholog of the bacterial and archaeal RecJ family nucleases. At least one RecJ homolog was identified in all sequenced archaeal genomes, with the single exception of Caldivirga maquilingensis. These proteins include previously unnoticed remote RecJ homologs with inactivated DHH domain in Thermoproteales. Combined with phylogenetic tree reconstruction of diverse eukaryotic, archaeal and bacterial DHH subfamilies, this analysis yields a complex scenario of RecJ family evolution in Archaea which includes independent inactivation of the nuclease domain in Crenarchaeota and Halobacteria, and loss of this domain in Methanococcales.ConclusionsThe archaeal complex of a CDC45/RecJ homolog, MCM and GINS is homologous and most likely functionally analogous to the eukaryotic CMG complex, and appears to be a key component of the DNA replication machinery in all Archaea. It is inferred that the last common archaeo-eukaryotic ancestor encoded a CMG complex that contained an active nuclease of the RecJ family. The inactivated RecJ homologs in several archaeal lineages most likely are dedicated structural components of replication complexes.ReviewersThis article was reviewed by Prof. Patrick Forterre, Dr. Stephen John Aves (nominated by Dr. Purificacion Lopez-Garcia) and Prof. Martijn Huynen.For the full reviews, see the Reviewers' Comments section.

Multiple effects of honokiol on the life cycle of hepatitis C virus

Honokiol, a small active molecular compound extracted from magnolia, has recently been shown to inhibit hepatitis C virus (HCV) infection in vitro. This study further characterized aspects of the HCV lifecycle affected by the antiviral functions of honokiol. The influence of honokiol on HCV infection, entry, translation and replication was assessed in Huh-7.5.1 cells using cell culture-derived HCV (HCVcc), HCV pseudo-type (HCVpp) and sub-genomic replicons. Honokiol had strong antiviral effect against HCVcc infection at non-toxic concentrations. Combined with interferon-α, its inhibitory effect on HCVcc was more profound than that of ribavirin. Honokiol inhibited the cell entry of lentiviral particles pseudo-typed with glycoproteins from HCV genotypes 1a, 1b, and 2a, but not of the vesicular stomatitis virus. It had inefficient activity on HCV internal ribosome entry site (IRES)-translation at concentrations with significant anti-HCVcc effects. The expression levels of components of replication complex, NS3, NS5A and NS5B, were down-regulated by honokiol in a dose-dependent manner. It also inhibited HCV replication dose dependently in both genotypes 1b and 2a sub-genomic replicons. Honokiol inhibits HCV infection by targeting cell entry and replication and, only at a concentration >30μM, IRES-mediated translation of HCV life cycle. Based on its high therapeutic index (LD(50) /EC(90) =5.4), honokiol may be a promising drug for the treatment of HCV infection.

Involvement of Creatine Kinase B in Hepatitis C Virus Genome Replication through Interaction with the Viral NS4A Protein

Persistent infection with hepatitis C virus (HCV) is a major cause of chronic liver diseases. The aim of this study was to identify host cell factor(s) participating in the HCV replication complex (RC) and to clarify the regulatory mechanisms of viral genome replication dependent on the host-derived factor(s) identified. By comparative proteome analysis of RC-rich membrane fractions and subsequent gene silencing mediated by RNA interference, we identified several candidates for RC components involved in HCV replication. We found that one of these candidates, creatine kinase B (CKB), a key ATP-generating enzyme that regulates ATP in subcellular compartments of nonmuscle cells, is important for efficient replication of the HCV genome and propagation of infectious virus. CKB interacts with HCV NS4A protein and forms a complex with NS3-4A, which possesses multiple enzyme activities. CKB upregulates both NS3-4A-mediated unwinding of RNA and DNA in vitro and replicase activity in permeabilized HCV replicating cells. Our results support a model in which recruitment of CKB to the HCV RC compartment, which has high and fluctuating energy demands, through its interaction with NS4A is important for efficient replication of the viral genome. The CKB-NS4A association is a potential target for the development of a new type of antiviral therapeutic strategy.

Interaction of TIA-1/TIAR with West Nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly

The West Nile virus minus-strand 3' terminal stem loop (SL) RNA was previously shown to bind specifically to cellular stress granule (SG) components, T cell intracellular antigen-1 (TIA-1) and the related protein TIAR. In vitro TIAR binding was 10 times more efficient than TIA-1. The 3'(-)SL functions as the promoter for genomic RNA synthesis. Colocalization of TIAR and TIA-1 with the viral replication complex components dsRNA and NS3 was observed in the perinuclear regions of West Nile virus- and dengue virus-infected cells. The kinetics of accumulation of TIAR in the perinuclear region was similar to those of genomic RNA synthesis. In contrast, relocation of TIA-1 to the perinuclear region began only after maximal levels of RNA synthesis had been achieved, except when TIAR was absent. Virus infection did not induce SGs and progressive resistance to SG induction by arsenite developed coincident with TIAR relocation. A progressive decrease in the number of processing bodies was secondarily observed in infected cells. These data suggest that the interaction of TIAR with viral components facilitates flavivirus genome RNA synthesis and inhibits SG formation, which prevents the shutoff of host translation.

West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis

Using West Nile virus strain Kunjin virus (WNV(KUN)) as a model system for flavivirus replication, we showed that the virus replication complex (RC) is associated with the dsRNA template located in induced membranes only in the cytoplasm. In this report we established for the first time that the RNA-dependent RNA polymerase NS5 is located in flavivirus-induced membranes, including the site of viral RNA replication. We found no evidence for nuclear localization of the essential RC components NS5 and its dsRNA template for WNV(KUN) or the closely related WNV strain Sarafend, by immuno-electron microscopy or by immunofluorescence. Metabolic radiolabelling with [(32)P]orthophosphate revealed that WNV(KUN) NS5 was phosphorylated and this was confirmed by Western blotting with antibodies specific for phosphorylated serine and threonine only. These observations of a cytoplasmic location for the WNV polymerase and its phosphorylation state correspond to the characteristics of the hepatitis C virus RNA polymerase NS5B.

Functional Interaction of Hepatitis C Virus NS5B with Nucleolin GAR Domain

Hepatitis C Virus (HCV) non-structural proteins are major components of replication complex that is modulated by several host factors. We previously reported that nucleolin, a representative nucleolar marker, interacts with the NS5B through two separated sequences, amino acids (aa) 208-214 and 500-506, and that W208 in the former stretch is essential for both nucleolin-binding and HCV replication. Here we evaluated the role of the latter stretch aa 500-506 of WRHRARS in nucleolin-binding and HCV replication scanned by alanine-substituted clustered mutant (cm) or point mutant (pm). One tryptophan and three arginine residues in the sequence were found to be essential both for nucleolin-binding in vivo and HCV replication detected with a HCV subgenomic replicon transfected into Huh7 cells. NS5B-binding of nucleolin was further delineated by truncation and clustered mutants of nucleolin. Arginine-glycine-glycine (RGG) repeat in the Glycine arginine rich (GAR) domain were defined to be indispensable for NS5B-binding immunologically detected in in vivo and in vitro although short internal-truncations of RGG repeat are tolerable for NS5B-binding. These results indicate that nucleolin is a critical host factor for HCV replication through the direct interaction between W208 and several residues at the sequence, aa 500-505, of NS5B, and the long-turn motif including RGG repeat at nucleolin C-terminal.

Subproteomic study of hepatitis C virus replicon reveals Ras-GTPase-activating protein binding protein 1 as potential HCV RC component

Hepatitis C virus (HCV) RNA synthesis takes place on a detergent resistant membrane (DRM) structure. To identify potential cellular proteins related to HCV replication complexes (RC), we purified DRMs from HCV subgenomic replicon cells and its parental Huh7 cells. The proteins of DRM fractions were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Comparing with parental Huh7 cells, 60 proteins were up-regulated while 14 proteins were down-regulated in HCV replicon cells. Ras-GTPase-activating protein binding protein 1 (G3BP1), one of the elevated proteins, was found to be associated with HCV NS5B and knockdown of G3BP1 by siRNA in HCV replicon cells significantly reduced HCV replication, which may indicate it a potential component of HCV RC. These results suggest that HCV viral gene and proteins may regulate the presence of host cellular proteins in DRM, ensure appropriate concentrations of replication components, and hence control the rates or efficiencies of HCV replication.

An N-terminal amphipathic helix in HCV NS4B mediates membrane association and correct localization of HCV replication complex components

An N-terminal amphipathic helix in HCV NS4B mediates membrane association and correct localization of HCV replication complex components

An N-Terminal Amphipathic Helix in Hepatitis C Virus (HCV) NS4B Mediates Membrane Association, Correct Localization of Replication Complex Proteins, and HCV RNA Replication

Like other positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its RNA in association with host cell cytoplasmic membranes. Because of its association with such membranes, NS4B, one of the virus's nonstructural proteins, may play an important role in this process, although the mechanistic details are not well understood. We identified a putative N-terminal amphipathic helix (AH) in NS4B that mediates membrane association. Introduction of site-directed mutations designed to disrupt the hydrophobic face of the AH abolishes the AH's ability to mediate membrane association. An AH in NS4B is conserved across HCV isolates. Completely disrupting the amphipathic nature of NS4B's N-terminal helix abolished HCV RNA replication, whereas partial disruption resulted in an intermediate level of replication. Finally, immunofluorescence studies revealed that HCV replication complex components were mislocalized in the AH-disrupted mutant. These results identify a key membrane-targeting domain which can form the basis for developing novel antiviral strategies.

DNMT1 is a Component of a Multiprotein DNA Replication Complex

DNA methylation is a major determinant of epigenetic inheritance and plays an important role in genome stability. The accurate propagation of DNA methylation patterns with cell division requires that methylation be closely coupled to DNA replication, however the precise molecular determinants of this interaction have not been defined. In the present study, we show that the predominant DNA methyltransferase species in somatic cells, DNMT1, is a component of a multiprotein DNA replication complex termed the DNA synthesome that fully supports semi-conservative DNA replication in a cell-free system. DNMT1 protein and activity were found to co-purify with the human DNA synthesome through a series of subcellular fractionation and chromatography steps, resulting in an enrichment of methyltransferase specific activity from two human cell lines. DNA methyltransferase activity co-eluted with in vitro replication activity and DNA polymerase a activity on sucrose density gradients suggesting that DNMT1 is a tightly bound, core component of the replication complex. The synthesome-associated pool of DNA methyltransferase exhibited both maintenance and de novo methyltransferase activity and the ratio of the two was similar to that observed in whole cell lysates and for recombinant DNMT1. These data indicate that interactions within the synthesome complex do not influence the intrinsic preference of DNMT1 for hemimethylated DNA, but suggest that newly replicated DNA may be subject to low level de novo methylation. The data indicate that DNA methylation is tightly coupled to replication through physical interaction of DNMT1 and core components of the replication machinery. The definition of the molecular interactions between DNMT1 and other proteins in the replication complex in normal and neoplastic cells will provide further insight into the regulation of DNA methylation and the mechanisms underlying the alteration of DNA methylation patterns during carcinogenesis.

Hepatitis C Virus (HCV) NS5A Binds RNA-dependent RNA Polymerase (RdRP) NS5B and Modulates RNA-dependent RNA Polymerase Activity

Hepatitis C virus (HCV) NS5B is RNA-dependent RNA polymerase (RdRP), the essential catalytic enzyme for HCV replication. Recently, NS5A has been reported to be important for the establishment of HCV replication in vitro by the adaptive mutations, although its role in viral replication remains uncertain. Here we report that purified bacterial recombinant NS5A and NS5B directly interact with each other in vitro, detected by glutathione S-transferase (GST) pull-down assay. Furthermore, complex formation of these proteins transiently coexpressed in mammalian cells was detected by coprecipitation. Using terminally and internally truncated NS5A, two discontinuous regions of NS5A (amino acids 105-162 and 277-334) outside of the adaptive mutations were identified to be independently essential for the binding both in vivo and in vitro (Yamashita, T., Kaneko, S., Shirota, Y., Qin, W., Nomura, T., Kobayashi, K., and Mkyrakami, S. (1998) J. Biol. Chem. 273, 15479-15486). We previously examined the effect of His-NS5A on RdRP activity of the soluble recombinant NS5Bt in vitro (see Yamashita et al. above). Wild NS5A weakly stimulated at first (when less than 0.1 molar ratio to NS5B) and then inhibited the NS5Bt RdRP activity in a dose-dependent manner. The internal deletion mutants defective in NS5B binding exhibited no inhibitory effect, indicating that the NS5B binding is necessary for the inhibition. Taken together, our results support the idea that NS5A modulates HCV replication as a component of replication complex.