Production of infectious hepatitis C virus by using RNA polymerase I-mediated transcription.
In this study, we used an RNA polymerase I (Pol I) transcription system for development of a reverse genetics protocol to produce hepatitis C virus (HCV), which is an uncapped positive-strand RNA virus. Transfection with a plasmid harboring HCV JFH-1 full-length cDNA flanked by a Pol I promoter and Pol I terminator yielded an unspliced RNA with no additional sequences at either end, resulting in efficient RNA replication within the cytoplasm and subsequent production of infectious virions. Using this technology, we developed a simple replicon trans-packaging system, in which transient transfection of two plasmids enables examination of viral genome replication and virion assembly as two separate steps. In addition, we established a stable cell line that constitutively produces HCV with a low mutation frequency of the viral genome. The effects of inhibitors of N-linked glycosylation on HCV production were evaluated using this cell line, and the results suggest that certain step(s), such as virion assembly, intracellular trafficking, and secretion, are potentially up- and downregulated according to modifications of HCV envelope protein glycans. This Pol I-based HCV expression system will be beneficial for a high-throughput antiviral screening and vaccine discovery programs.
Masaki
Takahiro
T
Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan.
Suzuki
Ryosuke
R
Saeed
Mohsan
M
Mori
Ken-ichi
K
Matsuda
Mami
M
Aizaki
Hideki
H
Ishii
Koji
K
Maki
Noboru
N
Miyamura
Tatsuo
T
Matsuura
Yoshiharu
Y
Wakita
Takaji
T
Suzuki
Tetsuro
T
D001709:Biotechnology / Q000379:methods*; D002460:Cell Line; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000821:virology; D006801:Humans; D012318:RNA Polymerase I / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins; D014779:Virus Replication / Q000235:genetics*
D014764:Viral Proteins; D012318:RNA Polymerase I