A reporter cell line for rapid and sensitive evaluation of hepatitis C virus infectivity and replication.
The human pathogen hepatitis C virus (HCV) is associated with chronic liver disease. The recent development of the cell culture infectious HCV (HCVcc) system has opened up avenues for detailed studies on the life cycle of the virus and its interaction with the host cell. Current methods to quantitate virus infectivity in cell culture are time-consuming and labor-intensive. This study describes the generation of a cell-based secreted alkaline phosphatase (SEAP) reporter assay to facilitate in vitro studies of HCV infection and replication. This assay is based on a novel reporter cell line stably expressing the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted alkaline phosphatase via a recognition sequence of the viral NS3/4A serine protease. The SEAP reporter from a similar construct has previously been shown to be released from the fusion protein and be secreted into the extracellular culture medium following cleavage by the viral NS3/4A protease. The reporter cell line enabled rapid and sensitive quantification of HCV infection and viral replication in cell culture. The utility of this system for investigating virus entry, and for high throughput screening of entry inhibitors and other antiviral compounds was demonstrated using several inter- and intra-genotypic chimeras of HCV.
Iro
Michaela
M
MRC Virology Unit, Institute of Virology, University of Glasgow, Glasgow, United Kingdom.
Witteveldt
Jeroen
J
Angus
Allan G N
AG
Woerz
Ilka
I
Kaul
Artur
A
Bartenschlager
Ralf
R
Patel
Arvind H
AH
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D018929:Cell Culture Techniques; D002460:Cell Line; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000254:growth & development / Q000472:pathogenicity*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013997:Time Factors; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000469:Alkaline Phosphatase; D002352:Carrier Proteins; D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus; C485184:enhanced green fluorescent protein