Functional analysis of hepatitis C virus envelope proteins, using a cell-cell fusion assay.
Hepatitis C virus (HCV) envelope proteins mediate the entry of virus into cells by binding to cellular receptors, resulting in fusion of the viral membrane with the host cell membrane and permitting the viral genome to enter the cytoplasm. We report the development of a robust and reproducible cell-cell fusion assay using envelope proteins from commonly occurring genotypes of HCV. The assay scored HCV envelope protein-mediated fusion by the production of fluorescent green syncytia and allowed us to elucidate many aspects of HCV fusion, including the pH of fusion, cell types that permit viral entry, and the conformation of envelope proteins essential for fusion. We found that fusion could be specifically inhibited by anti-HCV antibodies and by at least one peptide. We also generated a number of insertional mutations in the envelope proteins and tested nine of these using the fusion assay. We demonstrate that this fusion assay is a powerful tool for understanding the mechanism of HCV-mediated fusion, elucidating mutant function, and testing antiviral agents.
Kobayashi
Mariko
M
Department of Pathology, Columbia University Medical Center, 630 West 168th St., New York, NY 10032, USA.
Bennett
Michael C
MC
Bercot
Theodore
T
Singh
Ila R
IR
D000998:Antiviral Agents / Q000494:pharmacology; D002459:Cell Fusion / Q000379:methods*; D002460:Cell Line; D019084:Fluorescent Antibody Technique, Indirect; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence; D009154:Mutation; D011487:Protein Conformation; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D000998:Antiviral Agents; D014759:Viral Envelope Proteins; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins