Molecular Mechanism Of Hbv Replication Prompted By HBX Protein Through Regulating Cell Cycle In Primary Mouse Hepatocytes

Background and objective: The hepatitis B virus X protein (HBx), amultifunctional regulatory protein encoded by HBV, is known to be closelyrelated with HBV replication and the development of HBV-associatedhepatocellular carcinoma (HCC), but the specific regulatory mechanism isstill not clear. Recent studies in transformed or immortalized cell linesshowed that HBx is required for efficient HBV replication and theCOOH-terminal region of HBx protein plays a key role in this regulation.However, in normal hepatocytes, the pathway for regulation of HBVreplication and cell proliferation by HBx and its functional region isunclear. We have evaluated the effects of HBx protein as well as itstruncation mutants on cell cycle and HBV replication in normal primarycultured mouse hepatocytes, and their relationship, and have determinedthe main functional region of HBx protein, so as to reveal the mechanism of regulation of cell cycle and HBV replication by HBx protein.Methods:(1) Primary mouse hepatocytes in C57BL/6J mice were isolated andpurified by modified in situ two-step collagenase perfusion, and then werecultured in serum-free Williams' Medium E (WME) with varioussupplements. Liver-specific functions and differentiated properties wereconfirmed by PAS staining and biochemical techniques. The cell cyclemarkers, including p16, p21, cyclin D1, cyclin E and cyclin A, of freshlyisolated and primary cultured hepatocytes were monitored by Western blotanalysis.(2) Primary cultured mouse hepatocytes were transiently transfectedwith pSI-HA-X, pSI-HA-X^1-101and pSI-HA-X^43-154 which respectivelyexpresses HBx protein and its truncations. The expression of HBx proteinwas detected by IP/Western blot analysis, and the cell cycle markersdescribed above were detected by Western blot analysis.(3) Primary cultured mouse hepatocytes were transiently transfectedwith pGEM-HBV and pGEM-HBVâ–³X respectively encoding the wholeHBV genome (payw1.2) and the same genome with a deletion mutation inthe HBx ORF, the latter was co-transfected with pSI-HA-X, pSI-HA-X^1-101and pSI-HA-X^43-154. Then the expression of HBx protein was detected byIP/Western blot analysis. The cell cycle markers described above and HBcprotein encoding by HBV were detected by Western blot analysis. The level of HBV replication was evaluated by Southern blot analysis and real-timePCR.(4) Primary cultured mouse hepatocytes were transiently transfectedwith pGEM-HBV and pGEM-HBVâ–³X respectively, and wereco-transfected with miRNA expression plasmids for mRNA interference ofp53. The efficacy of knockdown of p53was evaluated by IP/Western blotanalysis. The cell cycle markers described above and HBc protein encodingby HBV were detected by Western blot analysis. The level of HBVreplication was evaluated by Southern blot analysis and real-time PCR.Results:(1) Both liver-specific functions and differentiated properties,including expression of glycogen, albumin and urea, was generallymaintained in the cultured primary mouse hepatocytes. The culturedprimary mouse hepatocytes preserve a quiescent cell cycle status (G0phase)indistinguishable from that of the freshly isolated hepatocytes.(2) Full-length HBx protein, COOH-terminally truncated mutationHBx1-101and NH2-terminally truncated mutation HBx43-154were able to bedetected by IP/Western blot analysis in transfected primary mousehepatocytes. HBx43-154was at approximately the same level as full-lengthHBx, but the level of HBx1-101was significantly lower than that of theformer two. Full-length HBx protein and HBx43-154decreased the level ofp16protein, increased both the level of p21, cyclin D and cyclin E, and didn't affect the level cyclin A, while HBx1-101had no effect on those cellcycle markers even when the expression of HBx1-101was enhanced byincreasing the amount of pSI-HA-X^1-101.(3) Cells transfected with pGEMHBV demonstrated decreased level ofCDK inhibitor p16, elevated levels of p21, cyclin D and cyclin E andunchanged level of cyclin A in comparison to pGEMHBVâ–³X-transfectedcells, and the level of capsid-associated HBV DNA inpGEMHBV-transfected cells was significantly higher than that inpGEMHBVâ–³X-transfected cells. Cell cycle markers and capsid-associatedHBV DNA in pGEM-HBVâ–³X-transfected cells were restored to the samelevels as in pGEM-HBV-transfected cells after the deleted HBx protein wasreconstructed by full-length HBx and HBx43-154, respectively. However, thelevels of cell cycle markers and capsid-associated HBV DNA inpGEM-HBVâ–³X-transfected cells did not change even when theexpression of HBx1-101was enhanced to the same level as full-length HBxand HBx43-154by increasing the amount of pSI-HA-X^1-101. The expressionof HBc protein encoded by HBV was generally identical in each group.(4) In pGEM-HBVâ–³X-transfected hepatocytes, down-regulation ofp53resulted in a decrease in the level of p16protein but an increase in thelevels of cyclin D, cyclin E and cyclin A, with no change in the level of p21.and the level of HBV replication was found to be increased to a certainextent, while still dramatically lower than the wild-type level of viral DNA from pGEM-HBV. In pGEM-HBV-transfected hepatocytes, knockdown ofp53could bring about little or no alteration in the levels of cell cycleproteins and viral replication. The expression of HBc protein encoded byHBV was generally identical in each group.Conclusion: In primary mouse hepatocytes retaining a quiescent cellcycle state, HBx protein can induce cells to enter G1phase from G0phaseby repressing the expression of p16and increasing the levels of cyclin Dand cyclin E, and arrest cells in G1phase by increasing p21protein tostimulate HBV replication. HBx is able to override the promotion of cellcycle progression to S phase as a result of knockdown of p53protein byincreasing p21protein to block cells in G1phase to stimulate HBVreplication. In primary cultured mouse hepatocytes, HBx protein plays akey role in maintaining a maximal level of HBV replication, and theCOOH-terminal region of HBx is important for its promotion of HBVreplication through cell cycle regulation.