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. 2010 Feb;84(4):1666-73.
doi: 10.1128/JVI.02043-09. Epub 2009 Dec 9.

Genetic analysis of the carboxy-terminal region of the hepatitis C virus core protein

Martina Kopp  1 , Catherine L Murray, Christopher T Jones, Charles M Rice
Affiliations

Genetic analysis of the carboxy-terminal region of the hepatitis C virus core protein

Martina Kopp et al. J Virol. 2010 Feb.

Abstract

Hepatitis C virus (HCV) is a liver-tropic pathogen with severe health consequences for infected individuals. Chronic HCV infection can progress to cirrhosis and hepatocellular carcinoma and is a leading indicator for liver transplantation. The HCV core protein is an essential component of the infectious virus particle, but many aspects of its role remain undefined. The C-terminal region of the core protein acts as a signal sequence for the E1 glycoprotein and undergoes dual processing events during infectious virus assembly. The exact C terminus of the mature, virion-associated core protein is not known. Here, we performed genetic analyses to map the essential determinants of the HCV core C-terminal region, as well as to define the minimal length of the protein that can function for infectious virus production in trans.

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Figures

FIG. 1.
FIG. 1.
Core mutant replication and infectious virus production. (A) RNA replication, as quantified by a Renilla luciferase assay, at 6 h (white) and 48 h (black) postelectroporation. (B) Infectious virus production at 48 h postelectroporation was measured by assay of Renilla luciferase activity in infected Huh-7.5 cell lysates. The dashed horizontal line represents background levels of luciferase activity. J6/JFH(p7-Rluc2A) residues changed to alanine or leucine are identified. ΔE1E2, J6/JFH(p7-Rluc2A)/ΔE1E2; GNN, J6/JFH(p7-Rluc2A)/GNN; RLU, relative light units. Means and standard errors of the means (SEM) of results from at least duplicate electroporations are shown.
FIG. 2.
FIG. 2.
Intracellular protein expression. Lysates harvested 48 h postelectroporation were analyzed by Western blotting. Residues mutated to alanine are identified. WT, wild type [J6/JFH(p7-Rluc2A)]. Blots are representative of results of at least duplicate analyses from independent electroporations.
FIG. 3.
FIG. 3.
Noninfectious particles are not released by core mutant genomes. (A) Viral RNA release into the culture supernatant at 48 h postelectroporation. Means and SEM of results from at least duplicate electroporations are shown. (B) HCV core protein release into the culture supernatant at 48 h postelectroporation. Residues mutated to alanine are identified. Horizontal lines indicate background levels based on that of negative controls.
FIG. 4.
FIG. 4.
Intracellular infectious virus production by core mutant genomes. Cell lysates generated by multiple rounds of freeze-thaw treatment at 48 h postelectroporation were used to infect naïve Huh-7.5 cells in the presence of isotype control antibody (anti-IgG1) or the HCV-blocking antibody (anti-CD81). Residues mutated to alanine are identified. Means and SEM of results from at least duplicate electroporations are shown.
FIG. 5.
FIG. 5.
Intracellular localization of mutant core proteins. Huh-7.5 cells transfected with J6/JFH(p7-Rluc2A) and mutant genomes encoding an alanine substitution at residue 175 or 176 were analyzed by immunofluorescence staining and confocal microscopy. Cells were fixed at 72 h postelectroporation and stained for core protein (core) and lipid droplets (Bodipy).
FIG. 6.
FIG. 6.
Transcomplementation of a core deletion mutant by truncated core proteins. (A) Huh-7.5 cells were electroporated with a core deletion mutant, J6/JFH(p7-Rluc2A)/Δ57-160, alone (−) or together with a VEE virus replicon expressing truncated HCV core protein (VEE-C vectors). Core C-terminal amino acids are indicated. Infectious virus production at 72 h postelectroporation was measured by assay of Renilla luciferase activity in infected Huh-7.5 cells. Means and SEM from at least duplicate electroporations are shown. (B) Western blot analyses of cell lysates electroporated with VEE virus plasmids carrying truncations of core protein. The truncated residues of core protein are indicated.
FIG. 7.
FIG. 7.
Alignment of core protein C-terminal regions. Core amino acid sequences from H77 (genotype 1a), Con1 (genotype 1b), J6 and JFH-1 (genotype 2a), S52 (genotype 3a), ED43 (genotype 4a), SA13 (genotype 5a), and EUHK (genotype 6a) were aligned using ClustalW. The shaded bar at the bottom represents the infectivity phenotypes of the mutant viruses. Black, no infectious virus production; gray, reduced titers; white, wild-type titers. Numbers indicate amino acid positions, and dots represent residues identical to those in the consensus sequence.
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