A method for in vitro assembly of hepatitis C virus core protein and for screening of inhibitors.
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. We developed a simple and rapid in vitro assay in which the progress of assembly is monitored by measuring an increase in turbidity, thereby allowing the kinetics of assembly to be determined. Assembly is performed using a truncated HCV core (C1-82), containing the minimal assembly domain, purified from Escherichia coli. The increase in turbidity is linked to the formation of nucleocapsid-like particles (NLPs) in solution, and nucleic acids are essential to initiate nucleocapsid assembly under the experimental conditions used. The sensitivity of NLP formation to salt strongly suggests that electrostatic forces govern in vitro assembly. Mutational analysis of C1-82 demonstrated that it is the global positive charge of C1-82 rather than any specific basic residue that is important for the assembly process. Our in vitro assembly assay provides an easy and efficient means of screening for assembly inhibitors, and we have identified several inhibitory peptides that could represent a starting point for drug design.
Fromentin
Rémi
R
Centre de Recherche en Infectiologie, Université Laval, Qué., Canada QC G1V 4G2.
Majeau
Nathalie
N
Laliberté Gagné
Marie-Eve
ME
Boivin
Annie
A
Duvignaud
Jean-Baptiste
JB
Leclerc
Denis
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D019251:Nucleocapsid / Q000187:drug effects / Q000502:physiology; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D012492:Salts / Q000494:pharmacology; D055672:Static Electricity; D014758:Viral Core Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D019065:Virus Assembly / Q000187:drug effects
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral; D011994:Recombinant Proteins; D012492:Salts; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus