Hepatitis C virus non-structural 5B protein interacts with cyclin A2 and regulates viral propagation.
OBJECTIVE
Hepatitis C virus (HCV) requires host cellular proteins for its own propagation. To identify the cellular factors necessary for HCV propagation, we have recently screened the small interfering RNA (siRNA) library targeting cell cycle genes using cell culture grown HCV (HCVcc)-infected cells. In the current study, we have selected and characterized the gene encoding Cyclin A2 (CycA2). Deregulation of CycA2 has been implicated in many types of cancers, including hepatocellular carcinoma.


METHODS
The effects of CycA2 on HCV propagation were investigated by siRNA-mediated knockdown assay, in vitro and in vivo protein binding assays, luciferase reporter gene assay, and immunoblot assay.


RESULTS
We showed that siRNA-mediated depletion of CycA2 significantly inhibited HCV replication in both HCV subgenomic replicon cells and HCVcc-infected cells. Furthermore, HCV non-structural 5B (NS5B) specifically interacted with CycA2 in vitro and in vivo. Protein interaction was mediated through the cyclin box of CycA2 and the palm domain of NS5B. We further showed that R/HxL motif in the palm domain of HCV NS5B mediated protein interaction with CycA2 and this interaction was necessary for HCV replication. Moreover, we demonstrated that tylophorine, the natural plant product exerting a CycA2 inhibitory function, abrogated HCV replication.


CONCLUSIONS
HCV regulates CycA2 via NS5B protein for its own propagation. In addition, tylophorine may be a potential therapeutic agent for HCV.
National Research Laboratory of Hepatitis C Virus, Ilsong Institute of Life Science, Hallym University, Anyang 431-060, Republic of Korea.
Long V
LV
Pham
Huong T T
HT
Ngo
Yun-Sook
YS
Lim
Soon B
SB
Hwang
D000470:Alkaloids / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000473:pathology / Q000821:virology; D002460:Cell Line; D002478:Cells, Cultured; D056751:Cyclin A2 / Q000187:drug effects / Q000378:metabolism*; D057809:HEK293 Cells; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D007212:Indolizines / Q000494:pharmacology; D007668:Kidney / Q000473:pathology / Q000821:virology; D008113:Liver Neoplasms / Q000473:pathology / Q000821:virology; D010616:Phenanthrenes / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology*; D012324:RNA-Dependent RNA Polymerase
D000470:Alkaloids; D000998:Antiviral Agents; D056751:Cyclin A2; D007212:Indolizines; D010616:Phenanthrenes; D034741:RNA, Small Interfering; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C004218:tylophorine