Abstract
Hepatitis C virus (HCV) assembly is still poorly understood. It is thought that trafficking of the HCV core protein to the lipid droplet (LD) surface is essential for its multimerization and association with newly synthesized HCV RNA to form the viral nucleocapsid. We carried out a mapping analysis of several complete HCV genomes of all genotypes, and found that the genotype 2 JFH-1 core protein contained 10 residues different from those of other genotypes. The replacement of these 10 residues of the JFH-1 strain sequence with the most conserved residues deduced from sequence alignments greatly increased virus production. Confocal microscopy of the modified JFH-1 strain in cell culture showed that the mutated JFH-1 core protein, C10M, was present mostly at the endoplasmic reticulum (ER) membrane, but not at the surface of the LDs, even though its trafficking to these organelles was possible. The non-structural 5A protein of HCV was also redirected to ER membranes and colocalized with the C10M core protein. Using a Semliki forest virus vector to overproduce core protein, we demonstrated that the C10M core protein was able to form HCV-like particles, unlike the native JFH-1 core protein. Thus, the substitution of a few selected residues in the JFH-1 core protein modified the subcellular distribution and assembly properties of the protein. These findings suggest that the early steps of HCV assembly occur at the ER membrane rather than at the LD surface. The C10M-JFH-1 strain will be a valuable tool for further studies of HCV morphogenesis.
Highlights
The WHO has estimated that about 150 million people worldwide are chronically infected by hepatitis C virus (HCV) and are, at risk of developing cirrhosis and liver cancer
The core sequences were translated in silico and the protein sequences obtained were compared with three reference sequences: JFH-1 [2], J6CF [38] and Dj, a genotype 1 core protein that has been shown to self-assemble into HCV-like particles (HCV-LPs) at endoplasmic reticulum (ER) membranes [37]
Previous studies have shown that the residue in position 20 is located in the RNA-binding domain [42], the residues in positions 75, 81 and 91 are part of the putative homodimerization domain [12,43], and the last five residues— 145, 147, 151, 172 and 173—are present in the Domain 2 (D2) domain, which is structured into two αhelices, resulting in association with the membrane and lipid droplet (LD) [14]
Summary
The WHO has estimated that about 150 million people worldwide are chronically infected by hepatitis C virus (HCV) and are, at risk of developing cirrhosis and liver cancer. Since 2005, the JFH-1 strain, a genotype 2 virus, has been used to generate infectious HCV in cell culture (HCVcc) [2], making it possible to study the essential steps of the viral life cycle, including virus assembly and release, in vitro [3,4] These late stages of the viral cycle are thought to take place at an interface between the surface of the cytoplasmic lipid droplets (LDs) and the cytosolic side of the endoplasmic reticulum (ER) membranes [5,6,7,8]. Many other functions have been proposed for this protein [10], but its sequence and structure suggest that it plays key roles in viral RNA encapsidation and in the acquisition of membrane-anchored envelope glycoproteins during HCV budding [4,11] This protein is 191 amino-acids long and has three distinct domains. This suggests that the JFH-1 core protein may be less efficient for viral assembly and/or secretion than the J6 core protein present in the genotype 2 Jc1 chimera or other HCV core proteins present in various intergenotypic chimeras
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