Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles.
ABSTRACT Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) possesses multiple and diverse functions in RNA replication, interferon resistance, and viral pathogenesis. Recent studies suggest that NS5A is involved in the assembly and maturation of infectious viral particles; however, precisely how NS5A participates in virus production has not been fully elucidated. In the present study, we demonstrate that NS5A is a prerequisite for HCV particle production as a result of its interaction with the viral capsid protein (core protein). The efficiency of virus production correlated well with the levels of interaction between NS5A and the core protein. Alanine substitutions for the C-terminal serine cluster in domain III of NS5A (amino acids 2428, 2430, and 2433) impaired NS5A basal phosphorylation, leading to a marked decrease in NS5A-core interaction, disturbance of the subcellular localization of NS5A, and disruption of virion production. Replacing the same serine cluster with glutamic acid, which mimics the presence of phosphoserines, partially preserved the NS5A-core interaction and virion production, suggesting that phosphorylation of these serine residues is important for virion production. In addition, we found that the alanine substitutions in the serine cluster suppressed the association of the core protein with viral genome RNA, possibly resulting in the inhibition of nucleocapsid assembly. These results suggest that NS5A plays a key role in regulating the early phase of HCV particle formation by interacting with core protein and that its C-terminal serine cluster is a determinant of the NS5A-core interaction.
Article: Hepatitis C virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins.[show abstract] [hide abstract]
ABSTRACT: Hepatitis C virus (HCV) and triglyceride-rich very low-density lipoproteins (VLDLs) both are secreted uniquely by hepatocytes and circulate in blood in a complex. Here, we isolated from human hepatoma cells the membrane vesicles in which HCV replicates. These vesicles, which contain the HCV replication complex, are highly enriched in proteins required for VLDL assembly, including apolipoprotein B (apoB), apoE, and microsomal triglyceride transfer protein. In hepatoma cells that constitutively produce infectious HCV, HCV production is reduced by two agents that block VLDL assembly: an inhibitor of microsomal triglyceride transfer protein and siRNA directed against apoB. These results provide a possible explanation for the restriction of HCV production to the liver and suggest new cellular targets for treatment of HCV infection.Proceedings of the National Academy of Sciences 05/2007; 104(14):5848-53. · 9.68 Impact Factor
Article: Modulation of retinoid signaling by a cytoplasmic viral protein via sequestration of Sp110b, a potent transcriptional corepressor of retinoic acid receptor, from the nucleus.[show abstract] [hide abstract]
ABSTRACT: Hepatitis C virus (HCV) core protein (core) plays a significant role in the development of chronic liver diseases caused by HCV infection. We have discovered that the core sensitized all-trans-retinoic acid (ATRA)-induced cell death in MCF-7 cells. Activation of retinoic acid receptor alpha (RARalpha)-mediated transcription by the core was also seen in all the cell lines tested. By use of a yeast two-hybrid system, we identified Sp110b as a candidate for a core-interacting cellular factor. Although the function of Sp110b has remained unknown, we observed that Sp110b interacts with RARalpha and suppresses RARalpha-mediated transcription. These data suggest that Sp110b is a transcriptional cofactor negatively regulating RARalpha-mediated transcription. RNA interference-mediated reduction of endogenous Sp110b levels depressed the ability of the core to activate RARalpha-mediated transcription, suggesting an essential role for Sp110b in this pathway. The normal nuclear subcellular localization of Sp110b was altered by molecular interaction with the core to the cytoplasmic surface of the endoplasmic reticulum. This evidence suggests a model in which the core sequesters Sp110b from the nucleus and inactivates its corepressor function to activate RARalpha-mediated transcription. These findings likely describe a novel system in which a cytoplasmic viral protein regulates host cell transcription.Molecular and Cellular Biology 12/2003; 23(21):7498-509. · 5.53 Impact Factor
Article: The hepatitis C virus core protein interacts with NS5A and activates its caspase-mediated proteolytic cleavage.[show abstract] [hide abstract]
ABSTRACT: Viral proteins interact with one another during viral replication, assembly, and maturation. Systematic interaction assays of the hepatitis C virus (HCV) proteins using the yeast two-hybrid method have uncovered a novel interaction between core and NS5A. This interaction was confirmed by in vitro binding assays, and coimmunoprecipitation in mammalian cells. Core and NS5A are also colocalized in COS-7 cells. Interestingly, NS5A is cleaved to give specific-size fragments, when core is coexpressed in mammalian cells. Overexpression of core produced many dying and rounded cells and effects such as DNA laddering and the truncation of poly(ADP-ribose) polymerase 1 (PARP1), both indicators of apoptosis. These observations led us to investigate the link between the induction of apoptosis by core and the cleavage of NS5A. The proteolysis of NS5A and these apoptotic events can be inhibited by caspase inhibitor, Z-VAD, indicating that core induces apoptosis and the cleavage of NS5A by caspases. In cells infected by the HCV, core may provide the intrinsic apoptotic signal, which produces truncated forms of NS5A. The biological function of core-NS5A interaction and the downstream effect of NS5A cleavage are discussed.Virology 12/2001; 290(2):224-36. · 3.35 Impact Factor