Screening, Identification And Expression Of Cellular Proteins Of Hepatocyte Binding To The Core Region Of Hepatitis C Virus RNA Genome

Objective and significanceHepatitis C virus (HCV) is one of causative pathogens of chronic hepatitis and liver cancer. However the molecular mechanisms underlying HCV replication and pathogenesis are poorly understood. A number of cellular factors from host cells may be involved in regulating HCV translation, replication and assembly by interacting with HCV RNA genome. These studies on HCV RNA-binding proteins were mainly focused on the 5'-UTR, 3'-UTR and negative strand of HCV RNA genome. But the studies on cellular proteins binding to core region of HCV RNA genome are still few. The objectives of this study were to screen, separate and identify cellular proteins, which could bind to core region of HCV RNA genome. It will help us to know about host cell-virus interactions, to regulate HCV translation and repulication at cellular level and to take measures in prevention and treatment of hepatitis C.Methods①The cDNA fragment of HCV core region was generated by the reverse-transcriptase-polymerase chain reaction (RT-PCR), and the plasmid pGEM-HCV was constructed to generate in vitro transcripts of core region of HCV RNA genome. Dig-labeled HCV C-RNA transcripts and unlabeled HCV C-RNA transcripts were obtained by in vitro transcription. After binding of HCV C-RNA and protein extracts from HepG2 cells, electrophoretic mobility shift assay (EMSA), Ultraviolet (UV) cross-linking experiment and competition analysis were performed to screen HepG2 cellular proteins, which interact with DIG-labeled transcripts of core region of HCV RNA genome. After electrophoresed on PAGE, proteins or RNA were transferred to NC membrane. DIG-labeled complexes were detected with anti-Digoxingenin-AP in order to find whether cellular proteins bound to HCV C-RNA or not.②The different HCV C-RNA transcripts in length were preparated by PCR, cloning and in vitro transcription. After binding of the different HCV C-RNA transcripts and protein extracts from HepG2 cells, UV cross-linking experiment was performed to identify binding region of HCV C-RNA. DIG labeled RNA-binding proteins were separated by immunoprecipitation with anti-DIG. By comparing SDS-PAGE's results with NC membrane's resluts, the proteins bands were excised from SDS-PAGE and were analyzed by MALDI-TOF-MS.③Total RNA was isolated from HepG2 cells, and the entire coding region of PGAB-B cDNA was obtained by RT-PCR. The cDNA fragment of PGAM-B was cloned into the pcDNA?3.1/V5-HisA, and the eukaryotic expression plasmid pcDNA-PGAM was constructed. The cDNA fragment of HCV core protein was obtained by PCR from plasmid pGEM-HCV and was cloned into pcDNA3.0 to construct the eukaryotic expression plasmid pcDNA-HCV. After identified by restriction endonuclease and sequencing,the eukaryotic expression plasmids were transfected into HepG2 cells respectively. The instantaneous expression of PGAM-His and HCV core protein were analyzed by immunocytochemical technique. Results①503 bp cDNA fragment of HCV core gene was obtained from HCV carrier. The plasmid pGEM-HCV was constructed and identified by PCR, restriction endonuclease and sequencing.②HCV cDNA sequence of core gene was compared with the known HCV gene sequence (AB092962.1) in GenBank. There were only 2 nucleotide differences between the two sequences, which located in position nt 67 (23aa K→E) and nt 177 (silent mutation).③By primary screening of EMSA, there were cellular proteins of HepG2 cells bingding to HCV C-RNA.④By screening of UV cross-linking experiment, there were several cellular proteins of HepG2 cells bingding to HCV C-RNA in vitro. The intensity of RNA binding protein bands increased with increasing amounts of cell extracts. The binding of cellular proteins to Digoxin labeled HCV C-RNA was competed out in proportion to the increasing amount of unlabeled HCV C-RNA.⑤Three different HCV C-RNA transcripts (198nt, 306nt and 503nt) could bind to cellular proteins in vitro, but the binding band of HCV C198 RNA to cellular proteins was clearer and sharper than that of others.⑥After immunoprecipitated, four RNA binding proteins were detedted in NC membrane. The protein band of P30 was the clearest, and the protein amount was also the largest.⑦The P30 protein bands were excised from SDS-PAGE and analysed by MALDI-TOF-MS. The P30 protein was identified as PGAM-B (Phosphoglycerate mutase isozyme B).⑧760bp fragment of PGAM-B cDNA sequence was obtained by RT-PCR from HepG2 cells, and it was consistent with the known PGAM-B sequence (J04173) in GenBank.⑨After the eukaryotic expression plasmids pcDNA-PGAM and pcDNA-HCV were transfected into HepG2 cells, the fusion protein PGAM-His and HCV core protein were instantaneous expressed in some of cells. Conclusions①There are several cellular proteins could specifically bind to core region of HCV RNA genome in vitro.②The binding site of HCV C-RNA maybe located in 5'-terminal of core region of HCV RNA genome.③PGAM-B could specifically bind to the core region of HCV RNA genome in vitro. It suggested that PGAM-B maybe involved in translation and replication of HCV RNA by interacting with core region of HCV RNA genome.④PGAM-B and HCV core peotein couled instantaneous express in HepG2 cells.