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Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3, NS4A, and NS4B.

ABSTRACT: NS5A of the hepatitis C virus (HCV) is a highly phosphorylated protein involved in resistance against interferon and required most likely for replication of the viral genome. Phosphorylation of this protein is mediated by a cellular kinase(s) generating multiple proteins with different electrophoretic mobilities. In the case of the genotype 1b isolate HCV-J, in addition to the basal phosphorylated NS5A (designated pp56), a hyperphosphorylated form (pp58) was found on coexpression of NS4A (T. Kaneko, Y. Tanji, S. Satoh, M. Hijikata, S. Asabe, K. Kimura, and K. Shimotohno, Biochem. Biophys. Res. Commun. 205:320-326, 1994). Using a comparative analysis of two full-length genomes of genotype 1b, competent or defective for NS5A hyperphosphorylation, we investigated the requirements for this NS5A modification. We found that hyperphosphorylation occurs when NS5A is expressed as part of a continuous NS3-5A polyprotein but not when it is expressed on its own or trans complemented with one or several other viral proteins. Results obtained with chimeras of both genomes show that single amino acid substitutions within NS3 that do not affect polyprotein cleavage can enhance or reduce NS5A hyperphosphorylation. Furthermore, mutations in the central or carboxy-terminal NS4A domain as well as small deletions in NS4B can also reduce or block hyperphosphorylation without affecting polyprotein processing. These requirements most likely reflect the formation of a highly ordered NS3-5A multisubunit complex responsible for the differential phosphorylation of NS5A and probably also for modulation of its biological activities.

SUBMITTER: Koch JO

PROVIDER: S-EPMC104237 | biostudies-literature | 1999 Sep

REPOSITORIES: biostudies-literature

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Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3, NS4A, and NS4B.

Koch J O JO Bartenschlager R R

Journal of virology 19990901 9

NS5A of the hepatitis C virus (HCV) is a highly phosphorylated protein involved in resistance against interferon and required most likely for replication of the viral genome. Phosphorylation of this protein is mediated by a cellular kinase(s) generating multiple proteins with different electrophoretic mobilities. In the case of the genotype 1b isolate HCV-J, in addition to the basal phosphorylated NS5A (designated pp56), a hyperphosphorylated form (pp58) was found on coexpression of NS4A (T. Kan ...[more]

PMID: 10438800

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Project description:Replication of the genotype 2 hepatitis C virus (HCV) requires hyperphosphorylation of the nonstructural protein NS5A. It has been known that NS5A hyperphosphorylation results from the phosphorylation of a cluster of highly conserved serine residues (S2201, S2208, S2211, and S2214) in a sequential manner. It has also been known that NS5A hyperphosphorylation requires an NS3 protease encoded on one single NS3-5A polyprotein. It was unknown whether NS3 protease participates in this sequential phosphorylation process. Using an inventory of antibodies specific to S2201, S2208, S2211, and S2214 phosphorylation, we found that protease-dead S1169A mutation abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues measured, consistent with the role of NS3 in NS5A sequential phosphorylation. These effects were not rescued by a wild-type NS3 protease provided in trans by another molecule. Mutations (T1661R, T1661Y, or T1661D) that prohibited proper cleavage at the NS3-4A junction also abolished NS5A hyperphosphorylation and phosphorylation at all serine residues, whereas mutations at the other cleavage sites, NS4A-4B (C1715S) or NS4B-5A (C1976F), did not. In fact, any combinatory mutations that prohibited NS3-4A cleavage (T1661Y/C1715S or T1661Y/C1976F) abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues. In the C1715S/C1976F double mutant, which resulted in an NS4A-NS4B-NS5A fusion polyprotein, a hyperphosphorylated band was observed and was phosphorylated at all serine residues. We conclude that NS3-mediated autocleavage at the NS3-4A junction is critical to NS5A hyperphosphorylation at S2201, S2208, S2211, and S2214 and that NS5A hyperphosphorylation could occur in an NS4A-NS4B-NS5A polyprotein.IMPORTANCE For ca. 20 years, the HCV protease NS3 has been implicated in NS5A hyperphosphorylation. We now show that it is the NS3-mediated cis cleavage at the NS3-4A junction that permits NS5A phosphorylation at serines 2201, 2208, 2211, and 2214, leading to hyperphosphorylation, which is a necessary condition for genotype 2 HCV replication. We further show that NS5A may already be phosphorylated at these serine residues right after NS3-4A cleavage and before NS5A is released from the NS4A-5A polyprotein. Our data suggest that the dual-functional NS3, a protease and an ATP-binding RNA helicase, could have a direct or indirect role in NS5A hyperphosphorylation.

Project description:UnlabelledFlavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here, we used dengue virus serotype 2 (DENV-2) as a model to characterize viral NS4A-NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant NS4A and NS4B proteins directly bind to each other with an estimated Kd (dissociation constant) of 50 nM. Amino acids 40 to 76 (spanning the first transmembrane domain, consisting of amino acids 50 to 73) of NS4A and amino acids 84 to 146 (also spanning the first transmembrane domain, consisting of amino acids 101 to 129) of NS4B are the determinants for NS4A-NS4B interaction. Nuclear magnetic resonance (NMR) analysis suggests that NS4A residues 17 to 80 form two amphipathic helices (helix α1, comprised of residues 17 to 32, and helix α2, comprised of residues 40 to 47) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix α3 (residues 52 to 75) that transverses the ER membrane. In addition, NMR analysis identified NS4A residues that may participate in the NS4A-NS4B interaction. Amino acid substitution of these NS4A residues exhibited distinct effects on viral replication. Three of the four NS4A mutations (L48A, T54A, and L60A) that affected the NS4A-NS4B interaction abolished or severely reduced viral replication; in contrast, two NS4A mutations (F71A and G75A) that did not affect NS4A-NS4B interaction had marginal effects on viral replication, demonstrating the biological relevance of the NS4A-NS4B interaction to DENV-2 replication. Taken together, the study has provided experimental evidence to argue that blocking the NS4A-NS4B interaction could be a potential antiviral approach.ImportanceFlavivirus NS4A and NS4B proteins are essential components of the ER membrane-associated replication complex. The current study systematically characterizes the interaction between flavivirus NS4A and NS4B. Using DENV-2 as a model, we show that NS4A interacts with NS4B in virus-infected cells, in cells transiently expressing NS4A and NS4B proteins, or in vitro with recombinant NS4A and NS4B proteins. We mapped the minimal regions required for the NS4A-NS4B interaction to be amino acids 40 to 76 of NS4A and amino acids 84 to 146 of NS4B. NMR analysis revealed the secondary structure of amino acids 17 to 80 of NS4A and the NS4A amino acids that may participate in the NS4A-NS4B interaction. Functional analysis showed a correlation between viral replication and NS4A-NS4B interaction, demonstrating the biological importance of the NS4A-NS4B interaction. The study has advanced our knowledge of the molecular function of flavivirus NS4A and NS4B proteins. The results also suggest that inhibitors of the NS4A-NS4B interaction could be pursued for flavivirus antiviral development.

Project description:UnlabelledA lack of immunocompetent-small-primate models has been an obstacle for developing hepatitis C virus (HCV) vaccines and affordable antiviral drugs. In this study, HCV/GB virus B (GBV-B) chimeric virus carrying the major nonstructural proteins NS2 to NS4A (HCV NS2 to -4A chimera) was produced and used to infect common marmosets, since HCV NS2 to NS4A proteins are critical proteases and major antigens. Seven marmosets were inoculated intrahepatically with HCV NS2 to -4A chimera RNA for primary infection or intravenously injected with chimera-containing serum for passage infection. Three animals used as controls were injected with phosphate-buffered saline (PBS) or GBV-B, respectively. Six of seven HCV NS2 to -4A chimera-infected marmosets exhibited consistent viremia and one showed transient viremia during the course of follow-up detection. All six infected animals with persistent circulating viremia presented characteristics typical of viral hepatitis, including viral RNA and proteins in hepatocytes and histopathological changes in liver tissue. Viremia was consistently detected for 5 to 54 weeks of follow-up. FK506 immunosuppression facilitated the establishment of persistent chimera infection in marmosets. An animal with chimera infection spontaneously cleared the virus in blood 7 weeks following the first inoculation, but viral-RNA persistence, low-level viral protein, and mild necroinflammation remained in liver tissue. The specific antibody and T-cell response to HCV NS3 in this viremia-resolved marmoset was boosted by rechallenging, but no viremia was detected during 57 weeks of follow-up. The chimera-infected marmosets described can be used as a suitable small-primate animal model for studying novel antiviral drugs and T-cell-based vaccines against HCV infection.ImportanceHCV infection causes approximately 70% of chronic hepatitis and is frequently associated with primary liver cancer globally. Chimpanzees have been used as a reliable primate model for HCV infection, but ethical considerations have restricted their utility in biomedical research. GB virus B (GBV-B) is a flavivirus related to HCV. It can infect common marmosets, a New World small primate, and induces viral hepatitis similar to HCV infection in humans. To minimize differences between GBV-B and HCV, we generated HCV NS2 to -4A/GBV-B chimeric viruses and established a chimera-infected marmoset model. HCV NS2 to -4A chimera-infected marmosets provide a small-animal model for evaluating novel antiviral drugs targeting HCV NS3-NS4A protease and T-cell-based HCV vaccines.

Project description:UnlabelledFlavivirus 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.ImportanceNS3 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.

Dataset Information

Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3, NS4A, and NS4B.

Publications

Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3, NS4A, and NS4B.

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