A method for in vitro assembly of hepatitis C virus core protein and for screening of inhibitors

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 using a truncated HCV core (C1-82), containing the minimal assembly domain, purified from E. 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; we have identified several inhibitory peptides that could represent a starting point for drug design.

Identification of Basic Amino Acids at the N-Terminal End of the Core Protein That Are Crucial for Hepatitis C Virus Infectivity

“…These reports highlighted the involvement of numerous amino acids in the production of infectious virus. Other studies, using cell-free and protein expression systems, reported that the N-terminal region of the core protein is important for HCV nucleocapsid assembly (15,25,26) and core protein-RNA interaction (49,57). However, this region of the core protein has not been fully investigated in the context of the HCVcc system.…”

Peptide inhibitors of hepatitis C virus core oligomerization and virus production

“…Fromentin et al (2007) studied a residue 1-82 core construct to follow nucleocapsid formation (Majeau et al, 2004;Fromentin et al, 2007). Based on the discovery of a residue 1-84 self-associating fragment by the yeast twohybrid method applied to a library of fragments of the HCV polyprotein (Flajolet et al, 2000) and the decision to include the residue 82-106 'homotypic' domain that had been reported to be essential for core dimerization Nolandt et al, 1997), we prepared the longer, residue 1-106 N-terminal fragment.…”

“…For this report, we chose not to work on nucleocapsid assembly, given the considerable difficulty in selecting the right RNA size and composition, the right core sequence, the reaction conditions and the best method of characterization of the resulting HCV nucleocapsid (Boulant et al, 2005;Fromentin et al, 2007). Instead, we evaluated the influence of the inhibitor peptides on infectious virus production by direct individual addition to cells infected with HCV.…”

“…Surprisingly, these peptides did not originate from the residue 82-106 homotypic region. Unfortunately, the peptides' specificities were not verified with any control, nor was their effect on HCV production studied (Fromentin et al, 2007).…”

“…Genotype 5 differs at position 102 by glycine to serine and at position 106 from serine to asparagine, and genotype 6 differs at position 98 by a leucine to methionine substitution, confirming that core is extremely well conserved across the six major genotypes. Fromentin et al (2007) also used core-derived peptides that inhibited complex formation with RNAs of various origins. Surprisingly, these peptides did not originate from the residue 82-106 homotypic region.…”