Development of NS3/4A protease-based reporter assay suitable for efficiently assessing hepatitis C virus infection.
A cell culture system for the production of hepatitis C virus (HCV) whole virions has greatly accelerated studies of the virus life cycle and the discovery of anti-HCV agents. However, the quantification of the HCV titers in a whole-virus infection/replication system currently relies mostly on reverse transcription-PCR or immunofluorescence assay, which would be cumbersome for high-throughput drug screening. To overcome this problem, this study has generated a novel cell line, Huh7.5-EG(Delta4B5A)SEAP, that carries a dual reporter, EG(Delta4B5A)SEAP. The EG(Delta4B5A)SEAP reporter is a viral protease-cleavable fusion protein in which the enhanced green fluorescence protein is linked to secreted alkaline phosphatase (SEAP) in frame via Delta4B5A, a short peptide cleavage substrate for NS3/4A viral protease. This study demonstrates that virus replication/infection in the Huh7.5-EG(Delta4B5A)SEAP cells can be quantitatively indicated by measuring the SEAP activity in cell culture medium. The levels of SEAP released from HCV-infected Huh7.5-EG(Delta4B5A)SEAP cells correlated closely with the amounts of HCV in the inocula. The Huh7.5-EG(Delta4B5A)SEAP cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target multiple stages of the HCV life cycle. The Z'-factor of this assay ranged from 0.64 to 0.74 in 96-well plates, indicating that this reporter system is suitable for high-throughput screening of prospective anti-HCV agents.
Pan
Kao-Lu
KL
Department of Chemical Engineering, National Tsing-Hua University, Hsin-Chu, Taiwan, Republic of China.
Lee
Jin-Ching
JC
Sung
Hsing-Wen
HW
Chang
Teng-Yuang
TY
Hsu
John T-A
JT
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000998:Antiviral Agents / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D017930:Genes, Reporter; D016174:Hepacivirus / Q000187:drug effects / Q000502:physiology*; D057166:High-Throughput Screening Assays; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000502:physiology*; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000469:Alkaline Phosphatase; D000998:Antiviral Agents; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and 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