Research On The Mechanism Of Host Restrictive Factor SAMHD1 On HBV Replication In Hepatic Cell

Objective: Hepatitis B virus is a DNA virus that replicates in reverse transcription.SAMHD1 is an interferon inducible triphosphohy-drolase which inhibits HBV replication by reducing the intracellular dNTP levels.However,whether its restriction activity is associated with its phosphorylation and how HBV regulate its phosphorylation in hepatoma cells are less unexplored.So we mainly discussed the relationship of the interaction between host restriction factor SAMHD1 and HBV:On one hand we show that as an interferon induced gene,SAMHD1 iInhibits HBV replication in the nucleus,its inhibitory effect depends on the activity of dNTPase.On the other hand,it elucidated the relationship between the phosphorylation of SAMHD1 and its antiviral effect,further screening and identificating the cell cycle kinase CDK2 kinase in liver cells had regulated the phosphorylation of SAMHD1 and viral replication,and the molecular mechanism of CDK2 directly acting on SAMHD1 in hepatocytes was confirmed by Co-IP and other methods.Research results help to clarify the host host restrictive factor SAMHD1 antiviral mechanism and raise the cell cycle HBV induced cell cycle enzyme CDK2 kinase phosphorylation SAMHD1,antagonizing its antiviral mechanism,and as a new antiviral targets for CDK2-SAMHD1 screening of new drugs to lay the foundation for the treatment of HBV.Methods: The first part: Study on the mechanism of SAMHD1 inhibiting HBV replication:(1)In hepatocytes,SAMHD1 is an interferon induced gene:The cell total RNA and protein were extracted after treated with interferon ?????.The RNA levels and protein were detected by qPCR and Western blot respectively.(2): HBV transfection replication model: The expression plasmid of SAMHD1 and HBV were Co-transfected in Huh7.0 cells,effect of exogenous SAMHD1 on HBV replication was determined by Southern blot.Meanwhile,effect of knocking down endogenous SAMHD1 on virus replication was determined by Southern blot.(3): SAMHD1 inhibit HBV replication is dependent on deoxynucleotide triphosphate hydrolase activity: SAMHD1 dNTPase active site mutantion was constructed by site mutation,RNA enzyme active site and nuclear localization signal site mutants,also on the HBV transfection replication model,effect of SAMHD1 different mutations on viral replication were determined by Southern blot,and changes of HBs Ag of supernatant secretion by ELISA;the exogenous and endogenous SAMHD1 position in liver cells by immunofluorescence and whether the antiviral effect depends on its nuclear localization signal by Southern blot.The second part: The effect of CDK2 phosphorylates SAMHD1 antagonized its restriction on HBV replication: Different phosphorylation site mutants of SAMHD1 were constructed,and the influence of these phosphorylation site mutants on HBV replication were detected by Southern blot.The phosphorylation level of SAMHD1,the replication of HBV,the cell cycle were detected by Western blot,Southern blot and flow cytometry respectively,after knocking down the cell cycle protein CDK1,CDK2,CDK6 in Huh7.0 cells.Then use the specific target cell cycle kinases inhibitors(CDK1 inhibitors,CDK2 inhibitors and CDK6 inhibitors)function in liver cell toward phosphorylation level of SAMHD1 the replication of HBV were detected.The interaction between SAMHD1 and Cyclin-dependent kinase(CDK)2 in Huh7.0 cells,and the interaction between SAMHD1 and CDK2 and HBV core protein were detected via Co-IP in 293 T.Then the inhibition of knockdown Cyclin E2 on SAMHD1 phosphorylation and HBV replication in cell model Huh7.0 cells and infection model NTCP-HepG2.0 were detected by Western blot,Southern blot,respectively.Results: The first part: The inhibition of SAMHD1 on HBV replication: Huh7.0 cells were treated with interferon ?????,the results showed that after being treated by interferon ???,transcription and translation levels of SAMHD1 obviously in dose-dependent increase.However,interferon ? showed no significant effect in the dose of 1000U/ml.Which indicates that SAMHD1 is mainly induced by type I interferon.The endogenous SAMHD1 was silenced in Huh7.0 cells,and the replication of HBV increased by 3.5 times compared with negative control.Kockdown the endogenous SAMHD1,analyse the the change of inhibition on HBV by interferon ???,the results show that: compared with control group,treated with interferon ?,SAMHD1 protein levels increased 2.16 times,and 76% reduction in the HBV replication;treated with interferon ?,the protein level of SAMHD1 increased 2.87 times,and HBV replication level decreased 81%.But after kocking down endogenous SAMHD1,interferon ? and interferon ? inhibition of HBV replication weakened obviously,which suggests that the role of interferon ? and interferon ? inhibition of HBV replication depend on the restrictive factors of endogenous SAMHD1.The overexpression of SAMHD1 in Huh7.0 cells and HepAD38 showed no significant toxicity to the cells.The cell localization of SAMHD1 was detected in Huh7.0 by immunofluorescence,we found that both the endogenous SAMHD1 and the over expression of SAMHD1 are localized in the nucleus,but the mutant protein of the missing nuclear localization signal(NLS)is located in the cytoplasm.Deletion of the SAMHD1 mutated protein D11-14 of NLS has lost its antiviral effect on HBV,indicating that the inhibitory effect of SAMHD1 on HBV depends on its nuclear localization.The second part: In HBV transfection-replication model Huh7.0 cells,the HBV replication plasmid and the SAMHD1 expression plasmid and its d NTPase activity mutation,nuclease mutation,and phosphorylation site mutation were performed.Compared with contrast,we found that the wild type and SAMHD1 T592V(dephosphorylation mutant)can inhibit HBV replication,however,dNTPase D207 N and activity of the mutant D137 N and T592 E phosphorylation lost its inhibition of HBV replication,indicating that the phosphorylation of the SAMHD1 protein,dNTPase activity and T592 sites is critical to its inhibition of HBV replication.When the double mutation D207N/T592 V cannot inhibit the replication of HBV,indicating that SAMHD1 dephosphorylation is related to its dNTPase activity.While the double mutation Q548A/T592 V can still inhibit the replication of HBV,indicating that the nucleases of SAMHD1 are not related to the antiviral effect.Deternmin the different phosphorylation mutants of SAMHD1(S18E,T21 E,T25E,S33 E,T592E)had no inhibitory effect on HBV only after the T592 site had mutated into phosphorylation.However,mutations in other phosphorylation sites,such as S18 E,T21E,T25 E,S33E and S93 E,still inhibit the activity of HBV.It indicated that the T592 phosphorylation site of SAMHD1 is an important site for antagonising antiviral activity.In Huh7.0 cells,the specific siRNA was transfected targeting CDK1,CDK2 and CDK6,the HBV replication plasmid was then expressed.The phosphorylated protein and the virus particle DNA of HBV were extracted.The results showed that the T592 phosphorylation of SAMHD1 and effect on the replication of HBV were not significantly affected by interfering CDK1 or CDK6.While after interfering CDK2,both the phosphorylation of the SAMHD1 protein T592 and the replication of HBV were significantly decreasing.It is indicated that compared to CDK1 and CDK6,CDK2 kinase is involved in the phosphorylation of SAMHD1 and negatively regulates its antiviral effect.Then the specific inhibitors of CDKs(CDK1,CDK2,CDK6)were treated in Huh7.0 cells,CDK2 inhibitor was found to inhibit the phosphorylation of SAMHD1 and inhibit the replication of HBV,while CDK1 and CDK6 inhibitors have no obvious effect on the phosphorylation level of SAMHD1 and the replication of HBV.SAMHD1 and CDK2 were transferred,and the interaction between SAMHD1 and CDK2 was found through Co-IP in Huh7.0 cells.In 293 T cells,the interaction between SAMHD1 and CDK2 was found,but HBV core protein was not involved in the activation form of SAMHD1/CDK2 by Co-IP.In the transfection replication model Huh7.0 cells and the infection model NTCP-HepG2 cells,cyclin E2 was found to participate in the activation form of SAMHD1/CDK2.Conclusion: In hepatic cells,endogenous SAMHD1 is regulated by interferon ? and ? to play its antiviral role.SAMHD1 inhibits HBV replication dependent on its dNTPase activity and cellular localization.The phosphorylation of the SAMHD1 protein T592 has negative antiviral effect.In hepatocytes,CDK2 kinase is involved in the regulation of phosphorylation of SAMHD1 and virus replication.CDK2 can have a direct effect on SAMHD1.Cyclin E2 is involved in the process of CDK2 phosphorylated SAMHD1 regulating virus replication.