Helper virus-independent trans-replication of hepatitis C virus-derived minigenome.
We have previously described a synthetic T7-driven cDNA minigenome containing the antisense sequence of luciferase gene and internal ribosome entry site of encephalomyocarditis virus flanked by 5'- and 3'-end sequences of hepatitis C virus (HCV) that contain cis-acting replication elements. Synthesis of minus-strand RNA from the artificial minigenome was determined by using Huh-7 cells harboring autonomously replicating HCV subgenome as a helper for provision of functional replication components. To further confirm and extend these studies, we investigated here whether the minigenome replication system could be reconstituted by transfection of naïve Huh-7 cells with plasmid expressing nonstructural (NS) proteins. Reporter assay and Northern blot analysis revealed that trans-expression of NS proteins from 3 to 5 resulted in high level of luciferase activity and synthesized minus-strand RNA. The analogous result was also obtained with the minigenome derived from HCV 2a, and both HCV 1b- and 2a-derived NS protein were able to support the chimeric minigenomes whose 5'- or 3'-end was replaced by the respective region of the heterologous virus. These results provide a basis for establishing the reverse genetic system that is helpful to study cis- and trans-acting factors involved in HCV RNA replication.
Zhang
Jing
J
Research and Development Center, FUSO Pharmaceutical Industries, LTD., 2-3-30 Morinomiya, Joto-ku, Osaka 536-8523, Japan. j-zhang@fuso-pharm.co.jp
Yamada
Osamu
O
Yoshida
Hiroshi
H
Sakamoto
Takashi
T
Araki
Hiromasa
H
Shimotohno
Kunitada
K
D045744:Cell Line, Tumor; D016679:Genome, Viral / Q000235:genetics*; D006378:Helper Viruses / Q000502:physiology; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D050506:Mutant Chimeric Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D050506:Mutant Chimeric Proteins; D014764:Viral Proteins; D012697:Serine Endopeptidases