Effect of hepatitis B virus X protein on the cell cycle of primary mouse hepatocytes / 中华肝脏病杂志

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>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.</p>