The Effect Of Hepatitis B Virus X Protein On The Cell Cycle Of Primary Mice Hepatocytes

Objective：To investigate the effect of HBV X protein on the cellcycle of primary mice hepatocytes in the context of HBV replication incultured primary mice hepatocytes, and to provide further evidence for themechanism of HBV X protein modulating cell cycle.Methods：Primary cultured mice hepatocytes were infected withpHBV or pHBV△X expressing plasmids. The levels of cell cycle proteinswere measured with Western blot. HBV DNA were analyzed by Southernblot and real-time PCR.Results：The levels of cell cycle proteins, including p16, cyclin D1,p21, cyclin E and cyclin A were not significantly different in freshlyisolated hepatocytes or cells collected24,48,72hours post-transfection.Cell cycle proteins extracted from hepatocytes which were transfected withpHBV or pHBV△X were collected48hours post-infection. The levels ofcyclin D1, p21, cyclin E were higher and the level of p16was lower inpHBV-transfected hepatocytes as compared to pHBV△X-transfectedhepatocytes,(t=15.713,22.897,14.680,-19.584respectively, P <0.05); however, the level of cyclin A was not affected (t=0.142，P>0.05）.Post-infection HBV DNA was extracted from hepatocytes72hours and assayed with Southern blot, the level of HBV DNA was higher inpHBV-transfected hepatocytes (rcDNA3288.336±448.011, dslDNA6458.318±182.163, ssDNA2760.613±393.561) as compared to pHBV△X-transfected hepatocytes (rcDNA515.721±62.530, dslDNA2122.228±28.347, ssDNA1632.013±207.021) and blank control group (P <0.05). Asimilar result was found in real-time PCR analysis,the copies were（987.50±47.80）and（303.67±33.94）per cell respectively (t=20.203, P<0.05).Conclusions：HBV X protein can affect the levels of cell cycle bymodulating cell cycle regulatory proteins which can induce quiescenthepatocytes to enter the G1phase of the cell cycle and stall in this phaseinstead of entering S phase to promote Hepatitis B Virus replication.