Screening cellular proteins binding to the core region of hepatitis C virus RNA genome with digoxin-labeled nucleic acids.
OBJECTIVE
To screen and identify cellular proteins binding to the core region of hepatitis C virus (HCV) RNA genome.


METHODS
The plasmid pHCV core was constructed to generate in vitro transcripts of the core region of HCV RNA genome. Ultraviolet (UV) cross-linking experiment and competition analysis were performed to screen HepG2 cellular proteins, which interact with digoxin-labeled transcripts of the core region of HCV RNA genome. RNA-binding proteins were separated by immunoprecipitation, analyzed by electrophoresis on SDS-PAGE and detected by immunoblotting with anti-digoxingenin-AP. After being excised from SDS-PAGE, the proteins bands were analyzed by MALDI-TOF-MS.


RESULTS
Several cellular proteins of hepG2 cell specifically bound to the core region of HCV RNA genome. The binding of cellular proteins to digoxin-labeled HCV core RNA was competed out in proportion to the increasing amount of unlabeled RNA. One of the HCV RNA-binding proteins was the B (brain) isozyme of human phosphoglycerate mutase (PGAM-B) identified by MALDI-TOF-MS.


CONCLUSIONS
PGAM-B could specifically bind to the core region of HCV RNA genome in vitro.
Su
Hai-Xia
HX
Department of Epidemiology, Fourth Military Medical University, Xi'an, China.
Xu
De-Zhong
DZ
Zhang
Yu-Hai
YH
Men
Ke
K
Zhao
Xiao-Ning
XN
Li
Duan
D
Zhang
Lei
L
Zhang
Jing-Xia
JX
Yan
Yong-Ping
YP
D001665:Binding Sites; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000737:chemistry; D004077:Digoxin / Q000737:chemistry*; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D010736:Phosphoglycerate Mutase / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014466:Ultraviolet Rays; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*
D003432:Cross-Linking Reagents; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; D004077:Digoxin; D010736:Phosphoglycerate Mutase