Development of plaque assays for hepatitis C virus-JFH1 strain and isolation of mutants with enhanced cytopathogenicity and replication capacity.
HCV culture in vitro results in massive cell death, which suggests the presence of HCV-induced cytopathic effects. Therefore, we investigated its mechanisms and viral nucleotide sequences involved in this effect using HCV-JFH1 cell culture and a newly developed HCV plaque assay technique. The plaque assay developed cytopathic plaques, depending on the titer of the inoculum. In the virus-infected cells, the ER stress markers, GRP78 and phosphorylated eIF2-alpha, were overexpressed. Cells in the plaques were strongly positive for an apoptosis marker, annexin V. Isolated virus subclones from individual plaque showed greater replication efficiency and cytopathogenicity than the parental virus. The plaque-purified virus had 9 amino acid substitutions, of which 5 were clustered in the C terminal of the NS5B region. Taken together, the cytopathic effect of HCV infection involves ER-stress-induced apoptotic cell death. Certain HCV genomic structures may determine the viral replication capacity and cytopathogenicity.
Sekine-Osajima
Yuko
Y
Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan.
Sakamoto
Naoya
N
Mishima
Kako
K
Nakagawa
Mina
M
Itsui
Yasuhiro
Y
Tasaka
Megumi
M
Nishimura-Sakurai
Yuki
Y
Chen
Cheng-Hsin
CH
Kanai
Takanori
T
Tsuchiya
Kiichiro
K
Wakita
Takaji
T
Enomoto
Nobuyuki
N
Watanabe
Mamoru
M
D019943:Amino Acid Substitution; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology; D045744:Cell Line, Tumor; D003588:Cytopathogenic Effect, Viral / Q000235:genetics; D000091342:Endoplasmic Reticulum Chaperone BiP; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes / Q000378:metabolism; D005840:Gentian Violet; D006360:Heat-Shock Proteins / Q000378:metabolism; D016174:Hepacivirus / Q000235:genetics / Q000254:growth & development* / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D007211:Indoles / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis; D012093:Replicon / Q000235:genetics; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D010948:Viral Plaque Assay / Q000379:methods*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D017304:Annexin A5; D000091342:Endoplasmic Reticulum Chaperone BiP; D005456:Fluorescent Dyes; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D007211:Indoles; D018832:Molecular Chaperones; D012367:RNA, Viral; D014157:Transcription Factors; D014764:Viral Proteins; C101003:ELF2 protein, human; C007293:DAPI; D016650:Fluorescein-5-isothiocyanate; D005840:Gentian Violet