Palmitoylation of Hepatitis C Virus Core Protein Is Important for Virion Production

Nathalie Majeau,Rémi Fromentin,Christian Savard,Marie Duval,Michel J Tremblay,Denis Leclerc

doi:10.1074/jbc.m109.018549

Abstract

Hepatitis C virus core protein is the viral nucleocapsid of hepatitis C virus. Interaction of core with cellular membranes like endoplasmic reticulum (ER) and lipid droplets (LD) appears to be involved in viral assembly. However, how these interactions with different cellular membranes are regulated is not well understood. In this study, we investigated how palmitoylation, a post-translational protein modification, can modulate the targeting of core to cellular membranes. We show that core is palmitoylated at cysteine 172, which is adjacent to the transmembrane domain at the C-terminal end of core. Site-specific mutagenesis of residue Cys(172) showed that palmitoylation is not involved in the maturation process carried out by the signal peptide peptidase or in the targeting of core to LD. However, palmitoylation was shown to be important for core association with smooth ER membranes and ER closely surrounding LDs. Finally, we demonstrate that mutation of residue Cys(172) in the J6/JFH1 virus genome clearly impairs virion production.

Highlights

Recent studies have indicated that assembly of Hepatitis C virus (HCV) particles occurs on endoplasmic reticulum (ER) membranes that are associated closely with lipid droplets (LD) [11]
HCV core protein as well as the replication complex are found in the detergent-resistant membrane (DRM) fraction, which is distinct from the classical lipid rafts (14 –16)
Because HCV core is targeted to different organelle membranes during the viral life cycle, we investigated whether posttranslational modification of core in the form of palmitoylation could be involved in this trafficking

Summary

EXPERIMENTAL PROCEDURES

Construction of pPIC3.5Kcore, pcDNA3.1core, and J6/JFH1 Mutants—Clones of Pichia pastoris expressing HCV core C (aa 1–191) of strain H77 (genotype 1a) and spp, a signal peptide peptidase of human cell origin, have been described previously [19]. The proteins were precipitated using methanol and chloroform and resuspended in 1.2 ml of biotin buffer (0.4% SDS, 120 mM NaCl, 0.16% Triton X-100, 5 mM EDTA, 0.16 mM biotin-HPDP 1ϫ inhibitor mixture, 50 mM Tris1⁄7HCl, pH 7.4) and incubated for 1 h at room temperature. The cells were washed with PBS and lysed in radioimmune precipitation assay buffer (50 mM Tris1⁄7HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1ϫ complete protease inhibitors). ER Extraction and Fractionation—For extraction of ER membranes, Huh7.5 cells infected with vaccinia T7 polymerase and transfected for 24 h with pCDNA3.1 expressing core or core mutant C172S were washed with ice-cold phosphate-buffered saline (PBS), scraped into homogenization buffer (250 mM sucrose, 1 mM EDTA, 1ϫ complete protease inhibitors, 10 mM Hepes-NaOH, pH 7.4) to be disrupted by Dounce homogenization and repeated passages through a fine syringe needle. The statistical significance of the results was defined by performing a one-way analysis of variance combined with Turkey’s post tests to compare all pairs of columns

RESULTS

Palmitoylation site prediction in HCV core protein

DISCUSSION