Roles of full-length and truncated hepatitis B virus X protein and of interactions with the host-encoded damaged DNA binding protein 1 in HBV replication.
- Author:
Xuan YANG
1
;
Song HE
;
Na LUO
;
Li LUO
;
Hao FAN
;
Qian GONG
Author Information
- Publication Type:Journal Article
- MeSH: DNA-Binding Proteins; metabolism; Hep G2 Cells; Hepatitis B Surface Antigens; metabolism; Hepatitis B e Antigens; metabolism; Hepatitis B virus; metabolism; physiology; Host-Pathogen Interactions; Humans; Protein Isoforms; metabolism; Trans-Activators; metabolism; Transfection; Virus Replication
- From: Chinese Journal of Hepatology 2013;21(6):446-451
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo investigate the roles of the hepatitis B virus (HBV)-encoded X protein (HBx), including the full-length and truncated isoforms, and in conjunction with the host-encoded damaged DNA binding protein 1 (DDB1) in HBV replication.
METHODSRecominant expression plasmids carrying the wild-type HBV genome (pGEM-HBV1.2) or with deletion of the full-length HBx protein (pHBV-deltaX), or carrying the full-length HBx protein (pSI-X) or the HBx1to101 (pSI-X1to101) or HBx43to154 (pSI-X43to154) isoforms were constructed for transfection into HepG2 cells. The pcDNA6.2-GW/EmGFP-miR (DDB1-miRNA) vector was constructed for silencing of the DDB1 gene in co-transfected HepG2 cells. At 72 h after transfections, DDB1 silencing was confirmed by western blot analysis and real-time quantitive reverse transcription PCR, HBV DNA copies number was assessed by real time PCR, and levels of hepatitis B surface antigen (HbsAg) and hepatitis B e antigen (HbeAg) were determined by ELISA. Differences between groups was statistically analyzed by single-factor analysis of variance and the t-test.
RESULTSTransfection with pHBV-deltaX led to reductions in DDB1 mRNA (to 52.74% of that in the wild-type pGEM-HBV1.2 transfected cells), HBV replication (to 55.49%), HBsAg level (48.05%), and HBeAg level (46.22%). Co-transfection with pSI-X or pSI-X43to154, but not with pSI-X1to101, restored the pHBV-deltaX-induced reductions in DDB1 mRNA, HBV replication, HBsAg and HBeAg to wild-type levels. The quantity of DDB1 mRNA was approximately parallel with the quantity of HBV DNA copies in all the HepG2 transfection groups.
CONCLUSIONThe COOH-terminal amino acids of HBx are required for HBV replication in hepatocytes, possibly involving the host-encoded DDB1 protein.