A recombinant replication-competent hepatitis C virus expressing Azami-Green, a bright green-emitting fluorescent protein, suitable for visualization of infected cells.
The hepatitis C virus (HCV) production system consists of transfecting the human hepatoma cell line Huh7 with genomic HCV RNA (JFH1). To monitor HCV replication by fluorescence microscopy, we constructed a recombinant HCV clone expressing Azami-Green (mAG), a bright green fluorescent protein, by inserting the mAG gene into the nonstructural protein 5A (NS5A) gene; the resultant clone was designated JFH1-hmAG. The Huh-7.5.1 (a subclone of Huh7) cells transfected with JFH1-hmAG RNA were found to produce cytoplasmic NS5A-mAG, as readily visualized by fluorescence microscopy, and infectious virus, as assayed with the culture supernatant, indicating that JFH1-hmAG is infectious and replication-competent. Furthermore, the replication of this virus was inhibited by interferon alpha in a dose-dependent manner. These results suggest that JFH1-hmAG is useful for studying HCV life cycle and the mechanism of interferon's anti-HCV action and for screening and testing new anti-HCV drugs.
Hou
Wei
W
China-Japan Joint Laboratory of Molecular Immunology and Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences, Chaoyang District, Beijing, China.
Aoki
Chie
C
Yu
Lijuan
L
Wen
Xianzi
X
Xue
Yinhuan
Y
Gao
Bin
B
Liu
Wenjun
W
Gao
George Fu
GF
Iwamoto
Aikichi
A
Kitamura
Yoshihiro
Y
D000595:Amino Acid Sequence; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods*; D014162:Transfection; D014779:Virus Replication / Q000187:drug effects
D016898:Interferon-alpha; D049452:Green Fluorescent Proteins