OBJECTIVE: To investigate the effect of hepatitis B virus (HBV) X protein (HBx) on host cell cycle and HBV replication using cultured primary mouse hepatocytes to gain further insights into the mechanism of HBx-mediated modulation of cell cycle. METHODS: Primary cultured mouse hepatocytes were transfected with HBx-expressing (pHBV) or HBx-selected (pHBV triangle X) plasmids, which were generated with sequences of the HBV ayw subtype 1.2 and included the green fluorescent protein (GFP) reporter gene. The levels of cell cycle proteins (p16, cyclin D1, p21, cyclin E and cyclin A) were measured with Western blotting, and HBV DNA was analyzed by Southern blotting and real-time PCR. RESULTS: The freshly isolated hepatocytes showed no significant differences in levels of cell cycle proteins. However, at 48 hours post-transfection, the levels of cyclin D1, p21 and cyclin E were significantly higher and the level of p16 was significantly lower in the pHBV-transfected hepatocytes than in the pHBV triangle X-transfected hepatocytes (t = 15.713, 22.897, 14.680, and -19.584, respectively, P less than 0.05). The level of cyclin A was not different between the two groups (t = 0.142, P more than 0.05). At 72 hours post-transfection, the level of HBV DNA was higher in pHBV-transfected hepatocytes (rcDNA: 3288.336+/-448.011; dslDNA: 6458.318+/-182.163; ssDNA: 2760.613+/-393.561) than in pHBV triangle X-transfected hepatocytes (rcDNA: 515.721+/-62.530; dslDNA: 2122.228+/-28.347; ssDNA: 1632.013+/-207.021) and in the blank (untransfected) control group (P less than 0.05). Real-time PCR analysis of HBV DNA copy number per cell confirmed these results, (pHBV-transfected: 987.50+/-47.80 vs. pHBV triangle X-transfected: 303.67+/-33.94; t = 20.203, P less than 0.05). CONCLUSIONS: The HBx protein can affect the levels of cell cycle proteins, which may induce quiescent hepatocytes to enter the G1 phase of the cell cycle and stay in this phase instead of entering the S phase, thereby promoting HBV intracellular replication.
OBJECTIVE: To investigate the effect of hepatitis B virus (HBV) X protein (HBx) on host cell cycle and HBV replication using cultured primary mouse hepatocytes to gain further insights into the mechanism of HBx-mediated modulation of cell cycle. METHODS: Primary cultured mouse hepatocytes were transfected with HBx-expressing (pHBV) or HBx-selected (pHBV triangle X) plasmids, which were generated with sequences of the HBV ayw subtype 1.2 and included the green fluorescent protein (GFP) reporter gene. The levels of cell cycle proteins (p16, cyclin D1, p21, cyclin E and cyclin A) were measured with Western blotting, and HBV DNA was analyzed by Southern blotting and real-time PCR. RESULTS: The freshly isolated hepatocytes showed no significant differences in levels of cell cycle proteins. However, at 48 hours post-transfection, the levels of cyclin D1, p21 and cyclin E were significantly higher and the level of p16 was significantly lower in the pHBV-transfected hepatocytes than in the pHBV triangle X-transfected hepatocytes (t = 15.713, 22.897, 14.680, and -19.584, respectively, P less than 0.05). The level of cyclin A was not different between the two groups (t = 0.142, P more than 0.05). At 72 hours post-transfection, the level of HBV DNA was higher in pHBV-transfected hepatocytes (rcDNA: 3288.336+/-448.011; dslDNA: 6458.318+/-182.163; ssDNA: 2760.613+/-393.561) than in pHBV triangle X-transfected hepatocytes (rcDNA: 515.721+/-62.530; dslDNA: 2122.228+/-28.347; ssDNA: 1632.013+/-207.021) and in the blank (untransfected) control group (P less than 0.05). Real-time PCR analysis of HBV DNA copy number per cell confirmed these results, (pHBV-transfected: 987.50+/-47.80 vs. pHBV triangle X-transfected: 303.67+/-33.94; t = 20.203, P less than 0.05). CONCLUSIONS: The HBx protein can affect the levels of cell cycle proteins, which may induce quiescent hepatocytes to enter the G1 phase of the cell cycle and stay in this phase instead of entering the S phase, thereby promoting HBV intracellular replication.