Emergence of mutation clusters in the HCV genome during sequential viral passages in Sip-L expressing cells.
Sip-L, a member of the Cupin superfamily, is a hepatic factor capable of supporting hepatitis C virus (HCV) replication in an otherwise non-permissive cell line. HCV-positive serum was used to infect Huh-7 and 293 cells stably expressing Sip-L. Using the culture medium of the infected cells as an infection source, sequential viral passages were carried out in both cell lines. Efficient viral passage was observed in 293-Sip-L cells but not in Huh-7-Sip-L cells. The viral concentrations in the culture medium increased gradually from less than 10(2) copies/mL to 5.3 x 10(4) copies/mL after 25 sequential passages in 293-Sip-L cells. Sequence analysis of the viral genomes obtained from both the initial and final inocula revealed emergence of mutation clusters in NS2, NS3, and NS5A coding regions. Immunofluorescence study revealed that only a small percentage of infected cells expressed a detectable level of viral protein. Caspase 3 activities in the infected cells increased progressively during the viral passages. In conclusion, perpetual propagation of HCV was achieved using Sip-L expressing cells, allowing for the development of mutation clusters in the genome. The mutant HCV can be used as an infection source to study the molecular mechanism of HCV replication.
Hwang
Der-Ren
DR
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Taipei, Taiwan.
Lai
Hsin-Yu
HY
Chang
Ming-Ling
ML
Hsu
John T A
JT
Yeh
Chau-Ting
CT
D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line / Q000378:metabolism / Q000821:virology; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D009154:Mutation; D012692:Serial Passage; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
D053148:Caspase 3; D020169:Caspases; D017361:Viral Nonstructural Proteins; C097010:NS2 protein, Hepatitis C virus; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C505407:CASP3 protein, human