The N-terminal half of the core protein of hepatitis C virus is sufficient for nucleocapsid formation

Abstract

The core (C) protein of hepatitis C virus (HCV) appears to be a multifunctional protein that is involved in many viral and cellular processes. Although its effects on host cells have been extensively discussed in the literature, little is known about its main function, the assembly and packaging of the viral genome. We have studied the in vitro assembly of several deleted versions of recombinant HCV C protein expressed in E. coli. We demonstrated that the 75 N-terminal residues of the C protein were sufficient to assemble and generate nucleocapsid-like particles (NLPs) in vitro. However, homogeneous particles of regular size and shape were observed only when NLPs were produced from at least the first 79 N-terminal amino acids of the C protein. This small protein unit fused to the endoplasmic reticulum-anchoring domain also generated NLPs in yeast cells. These data suggest that the N-terminal half of the C protein is important for formation of NLPs. Similarities between the HCV C protein and C proteins of other members of the Flaviviridae are discussed.

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

“…Particles 25 nm ± 7 nm in diameter and uniform in appearance were produced (Fig. 1D) as expected and as previously reported [18]. No particles were seen under EM in grids with core protein only (data not shown), consistent with the previously published observation that recombinant C1-82 is a soluble and monodispersed protein [22].…”

“…The region of the core protein corresponding to amino acids 1-82 has been shown to be the minimal domain required for assembly [18]; we have previously shown that C1-82 protein forms NLPs when tRNA is added to the protein [18]. Recombinant C1-82 protein was purified by affinity chromatography (Fig.…”

“…The expression and purification of the recombinant proteins were performed using a Ni NTA resin (Qiagen) as previously described [18]. HCV-C proteins were eluted in assembly buffer (1.7 mM magnesium acetate, 100 mM potassium acetate, 25 mM Hepes (pH 7.4), and 500 mM imidazol).…”

“…The N-terminal half (N-terminal 82 amino acids) of the protein is rich in positively charged residues (K, R). This domain is sufficient to trigger the formation of nucleocapsid-like particles (NLPs) in vitro when structured RNA is added to the purified protein [18].…”

Evidence for cellular uptake of recombinant hepatitis C virus non‐enveloped capsid‐like particles

“…It is possible that these recombinant capsids could be empty, or that they could contain cellular RNA transcripts or the specific transcript. Although other systems have been used as well to study assembly of the HCV core protein [24,25,27,[53][54][55][56], the baculovirus expression system described here allows for the study of capsid assembly in a versatile, cell-based system and for the partial purification of the derived naked capsid-like particles in sufficient quantities, which might be useful for potential applications ranging from basic virological studies to developments in biomedicine.…”