Robust production of infectious viral particles in Huh-7 cells by introducing mutations in hepatitis C virus structural proteins.
Recently, the characterization of a cell culture system allowing the amplification of an authentic virus, named hepatitis C virus cell culture (HCVcc), has been reported by several groups. To obtain higher HCV particle productions, we investigated the potential effect of some amino acid changes on the infectivity of the JFH-1 isolate. As a first approach, successive infections of naïve Huh-7 cells were performed until high viral titres were obtained, and mutations that appeared during this selection were identified by sequencing. Only one major modification, N534K, located in the E2 glycoprotein sequence was found. Interestingly, this mutation prevented core glycosylation of E2 site 6. In addition, JFH-1 generated with this modification facilitated the infection of Huh-7 cells. In a second approach to identify mutations favouring HCVcc infectivity, we exploited the observation that a chimeric virus containing the genotype 1a core protein in the context of JFH-1 background was more infectious than wild-type JFH-1 isolate. Sequence alignment between JFH-1 and our chimera, led us to identify two major positions, 172 and 173, which were not occupied by similar amino acids in these two viruses. Importantly, higher viral titres were obtained by introducing these residues in the context of wild-type JFH-1. Altogether, our data indicate that a more robust production of HCVcc particles can be obtained by introducing a few specific mutations in JFH-1 structural proteins.
Delgrange
David
D
CNRS-UMR 8161, IBL, Université de Lille I et Lille II, Institut Pasteur de Lille, 59021 Lille cedex, France.
Pillez
André
A
CNRS-UMR 8161, IBL, Université de Lille I et Lille II, Institut Pasteur de Lille, 59021 Lille cedex, France.
Castelain
Sandrine
S
Laboratoire de Virologie, Centre Hospitalier Universitaire-Hôpital Sud, 80054 Amiens cedex, France.|CNRS-UMR 8161, IBL, Université de Lille I et Lille II, Institut Pasteur de Lille, 59021 Lille cedex, France.
Cocquerel
Laurence
L
CNRS-UMR 8161, IBL, Université de Lille I et Lille II, Institut Pasteur de Lille, 59021 Lille cedex, France.
Rouillé
Yves
Y
CNRS-UMR 8161, IBL, Université de Lille I et Lille II, Institut Pasteur de Lille, 59021 Lille cedex, France.
Dubuisson
Jean
J
CNRS-UMR 8161, IBL, Université de Lille I et Lille II, Institut Pasteur de Lille, 59021 Lille cedex, France.
Wakita
Takaji
T
Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan.
Duverlie
Gilles
G
Laboratoire de Virologie, Centre Hospitalier Universitaire-Hôpital Sud, 80054 Amiens cedex, France.|CNRS-UMR 8161, IBL, Université de Lille I et Lille II, Institut Pasteur de Lille, 59021 Lille cedex, France.
Wychowski
Czeslaw
C
CNRS-UMR 8161, IBL, Université de Lille I et Lille II, Institut Pasteur de Lille, 59021 Lille cedex, France.
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002460:Cell Line; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D015678:Viral Structural Proteins / Q000235:genetics*
D012367:RNA, Viral; D015678:Viral Structural Proteins