Study Of Effects Of SAMHD1 On Hepatitis B Virus And Mechanism Of SAMHD1 Expression Induced By IFN-α

Background & Objective: SAMHD1(Sterile alpha motif and HD-domain containing protein 1) is a newly identified intracellular antiviral factor. By depleting the d NTPs pool of host cells to a low level that can not support the efficient synthesis of viral c DNA, it restricts replication of some retroviruses. Studies have shown that SAMHD1 can restrict the replication of retroviruses, DNA viruses and the retrotransposition of retroelements.However, HIV-2(human immunodeficiency virus-2) and SIV(simian immunodeficiency virus) have evolved Vpx to counteract SAMHD1-mediated restriction of retroviral infection. HBV(Hepatitis B virus) infection is a global public health problem. Our country is a country of high prevalence of hepatitis B virus infection. Hepatitis B virus experiences a process of reverse transcription in its life cycle akin to that of retroviruses.However, whether SAMHD1 restricts HBV replication by reducing d NTPs in liver cell lines is unknown.Interferons are commonly used as a kind of anti-viral drugs to treatment of hepatitis B. Interferons often induce the expression of antiviral proteins to inhibit viral replication. It is well-known that IFN-α may induce the expression of genes through the canonical and non-canonical pathway. The canonical IFN-α signaling pathway requires ISGF3 complex which consists of STAT1、STAT2 and IRF9. The non-canonical signaling pathway is STAT1-independent. So far, how SAMHD1 expression is up-regulated by IFN-α in liver cells is unclear. In the present study, we examined the role of SAMHD1 in HBV replication in liver cells and whether IFN-α could be induced SAMHD1 expression in liver cells.Methods Four liver cell lines Hep G2、LO2、BEL-7402 and SMMC-7721 were utilized in this study,the endogenous expression of SAMHD1 in these four liver cell lines were detected by Western blot. The endogenous expression of SAMHD1 was further confirmed by immunofluorescence. p SAMHD1 wt and p SAMHD1 HD/AA were transfected into BEL-7402 and SMMC-7721 cells respectively, cell viability was measured by MTT method. p HBVwt were co-transfected transiently into SMMC-7721 and BEL-7402 cells with p SAMHD1 wt, p SAMHD1 HD/AA respectively. 48 h later, exogenous SAMHD1 expression was detected by Western blot. p HBVwt were transiently co-transfected into SMMC-7721 cells with p RL-TK and p SAMHD1 wt, p SAMHD1 HD/AA respectively. 48 h later, the levels of HBs Ag and HBe Ag in cell supernatants were determined by ELISA,HBV core-associated DNA levels in the cell supernatants were measured by real time PCR.Hep G2 cells were transfected with p HBVwt and p HBV△X, SAMHD1 expression was detected by Western blot. SMMC-7721 and BEL-7402 cells were treated with IFN-α at different doses and times, then SAMHD1 expression were determined by Western blot and real time PCR. SMMC-7721 cells were transfected with STAT1、STAT2 and IRF9 si RNA respectively,after further stimulation with IFN-α, cells were harvested for Western blot and real time PCR.Results SAMHD1 expression is detectable in four liver cell lines of Hep G2、LO2、BEL-7402 and SMMC-7721 cells; Exogenous expression of SAMHD1 has no cytotoxic effect on liver cells; SAMHD1 restricts HBV replication in SMMC-7721,BEL-7402 cells respectively; HBV replication down-regulates SAMHD1 expression in Hep G2 cells; IFN-α treatment increased SAMHD1 expression in a time-dependent and dose-dependent manner in SMMC-7721 and BEL-7402 cells; IFN-α treatment increased SAMHD1 m RNA levels in a time-dependent manner in SMMC-7721 cells; STAT1、STAT2 and IRF9 si RNA treatment suppress the induction of SAMHD1expression by IFN-α in SMMC-7721 cells respectively.Conclusion 1. SAMHD1 restricts HBV replication in liver cell lines; 2. SAMHD1 expression is up-regulated by IFN-α in liver cells; 3. IFN-α induces SAMHD1 expression through ISGF3 complex in SMMC-7721 cells.