The Replacement of 10 Non-Conserved Residues in the Core Protein of JFH-1 Hepatitis C Virus Improves Its Assembly and Secretion.
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.
Inserm U966, Faculté de medicine, Université François Rabelais and CHRU de Tours, Tours, France.
Loïc
L
Etienne
Inserm U966, Faculté de medicine, Université François Rabelais and CHRU de Tours, Tours, France; Plate-forme des Microscopies, PPF ASB, Université François Rabelais and CHRU de Tours, Tours, France.
Emmanuelle
E
Blanchard
Inserm U966, Faculté de medicine, Université François Rabelais and CHRU de Tours, Tours, France.
Audrey
A
Boyer
Inserm U966, Faculté de medicine, Université François Rabelais and CHRU de Tours, Tours, France.
Virginie
V
Desvignes
Plate-forme des Microscopies, PPF ASB, Université François Rabelais and CHRU de Tours, Tours, France.
Julien
J
Gaillard
Inserm U966, Faculté de medicine, Université François Rabelais and CHRU de Tours, Tours, France.
Jean-Christophe
JC
Meunier
Inserm U966, Faculté de medicine, Université François Rabelais and CHRU de Tours, Tours, France; Plate-forme des Microscopies, PPF ASB, Université François Rabelais and CHRU de Tours, Tours, France.
Philippe
P
Roingeard
Inserm U966, Faculté de medicine, Université François Rabelais and CHRU de Tours, Tours, France; Plate-forme des Microscopies, PPF ASB, Université François Rabelais and CHRU de Tours, Tours, France.
Christophe
C
Hourioux
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D045744:Cell Line, Tumor; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000648:ultrastructure / Q000821:virology; D004847:Epithelial Cells / Q000648:ultrastructure / Q000821:virology; D015870:Gene Expression; D005822:Genetic Vectors; D016679:Genome, Viral; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D022781:Hepatocytes / Q000648:ultrastructure / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics; D014758:Viral Core Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly / Q000235:genetics*; D017201:Virus Shedding / Q000235:genetics*
D012367:RNA, Viral; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins