Direct visualization of hepatitis C virus-infected Huh7.5 cells with a high titre of infectious chimeric JFH1-EGFP reporter virus in three-dimensional Matrigel cell cultures.
Identification of the hepatitis C virus (HCV) JFH1 isolate enabled the development of infectious HCV cell culture systems. However, the relatively low virus titres and instability of some chimeric JFH1 reporter viruses restricts some uses of this system. We describe a higher-titre JFH1-EGFP reporter virus where the NS5A V3 region was replaced with the EGFP gene and adapted by serial passage in Huh7.5 cells. Six adaptive mutants were identified: one each in E2, P7 and NS4B, plus three in the NS5A region. These adaptive mutants increased the reporter virus titres to 1×10(6) immunofluorescent focus-forming units ml(-1), which is the highest titre of JFH1-EGFP reporter virus reported to our knowledge. This chimeric virus did not lose EGFP expression following 40 days of passage and it can be used to test the activity of HCV antivirals by measuring EGFP fluorescence in 96-well plates. Moreover, this reporter virus allows living infected Huh7.5 cells in Matrigel three-dimensional (3D) cultures to be visualized and produces infectious viral particles in these 3D cultures. The chimeric NS5A-EGFP infectious JFH1 reporter virus described should enable new studies of the HCV life cycle in 3D cell cultures and will be useful in identifying antivirals that interfere with HCV release or entry.
Department of Medicine, School of Medicine, University of Utah, Salt Lake City, UT 84132, USA.
Shuanghu
S
Liu
Department of Medicine, School of Medicine, University of Utah, Salt Lake City, UT 84132, USA.
Ren
R
Chen
Central Arkansas Veterans Healthcare System and University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.|Department of Medicine, School of Medicine, University of Utah, Salt Lake City, UT 84132, USA.
Curt H
CH
Hagedorn
D018929:Cell Culture Techniques; D002460:Cell Line; D003094:Collagen; D004338:Drug Combinations; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics / Q000254:growth & development*; D022781:Hepatocytes / Q000821:virology*; D006801:Humans; D007797:Laminin; D008969:Molecular Sequence Data; D011509:Proteoglycans; D012367:RNA, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling / Q000379:methods*; D019562:Viral Load; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*; D012324:RNA-Dependent RNA Polymerase
D003094:Collagen; D004338:Drug Combinations; D049452:Green Fluorescent Proteins; D007797:Laminin; D011509:Proteoglycans; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C485184:enhanced green fluorescent protein; C059485:matrigel; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase