A method for in vitro assembly of HCV core protein and for screening of inhibitors 一种HCV核心蛋白体外组装及抑制剂筛选方法.pdfVIP

NIH Public Access Author Manuscript Anal Biochem . Author manuscript; available in PMC 2008 July 1. N Published in final edited form as: I H Anal Biochem . 2007 July 1; 366(1): 37–45. - P A A A method for in vitro assembly of HCV core protein and for u t h o screening of inhibitors r M a Rémi Fromentin, Nathalie Majeau, Marie-Eve Laliberté Gagné, Annie Boivin, Jean-Baptiste n u Duvignaud, and Denis Leclerc s c Centre de Recherche en Infectiologie, Pav. CHUL, Université Laval, 2705 boul. Laurier, Québec, r i p (Qc), CANADA, G1V 4G2 t Abstract The assembly of hepatitis C virus (HCV) is not well understood. We investigated HCV nucleocapsid assembly in vitro and the role of electrostatic/hydrophobic interactions in this process. A simple and rapid in vitro assay was developed in which the progress of assembly is monitored by measuring an increase in turbidity, thus allowing the kinetics of assembly to be determined. Assembly is performed N I using a truncated HCV core (C1-82), containing the minimal assembly domain, purified from E. H - coli. The increase in turbidity is linked to the formation of nucleocapsid-like particles (NLPs) in P A solution, and nucleic acids are essential to initiate nucleocapsid assembly under the experimental A conditions used. The sensitivity of NLP formation to salt strongly suggests that electrostatic forces u govern in vitro assembly. Mutational analysis of C1-82 demonstrated that it is the global positive t h o charge of C1-82 rather than any specific basic residue that is important for the asse