Establishment of a Stable Cell Line Overexpressing Human SAMHD1 Gene and Its Antiviral Activity / 中国医科大学学报

Objective A lentivirus-mediated colon cancer cell line stably overexpressing human SAMHD1 gene was constructed to observe the effect of this gene on the ability of the cells to resist herpes simplex virus type 1 (HSV-1) infection. Methods p CDH-EF1-MCS-SAMHD1-T2 A-GFP recombinant plasmid was constructed using pCDH-EF1-MCS-T2 A-copGFP lentiviral vector. After confirming successful synthesis with sequencing, the recombinant plasmid and lentivirus packaging plasmids were co-transfected into 293 T cells. The pseudovirus solution was collected and concentrated, then human colon cancer cells were infected with the concentrated solution. The cell line overexpressing SAMHD1 gene was infected with HSV-1 in contrast to the control group, and the virus infection efficiency was assessed by Western blotting and immunofluorescence analyses. Results The pCDH-EF1-MCS-SAMHD1-T2 A-GFP recombinant plasmid was successfully constructed and verified by gene sequencing. Western blotting confirmed the overexpression of the SAMHD1 gene with higher levels of the gene in the transfected cells in contrast to control human colon cancer cell line. Western blotting and immunofluorescence showed that the cell line overexpressing human SAMHD1 gene could effectively inhibit the infection of HSV-1. Conclusion Lentivirus-mediated stable cell line overexpressing human SAMHD1 gene was effective in inhibiting HSV-1 replication and could help us to further investigate the function of SAMHD1.

Inhibitory effect of SAMHD1 on production of HBV DNA / 中国免疫学杂志

Objective:To study the expression levels of SAMHD1 in different liver cancer cells and the inhibition of HBV DNA production by SAMHD1. Methods:SAMHD1 mRNA and protein levels in Jurkat,THP-1,HepG2,HepG2. 2. 15 and Huh7 were detected by Real-time quantitative PCR and Western blot. SAMHD1 expression in HepG2. 2. 15 was increased by transfecting SAMHD1 over ex-pression vectors or was inhibited by transfecting SAMHD1 specific shRNA, and then HBV DNA levels were detected. The effect of different dNTP concentration on HBV DNA production was detected also. Results:SAMHD1 were highly expressed in liver cancer cells. Decreased SAMHD1 expression could enhance HBV DNA production by 2. 3-2. 8 times,and increased SAMHD1 expression could inhibit HBV DNA levels by 3 times(P<0. 01). Increased dNTP concentration could enhance HBV DNA production and counteract the inhibitory effect of SAMHD1. Conclusion:SAMHD1 can inhibit the production of HBV DNA in liver cancer cells by decreasing dNTP concentration.

Inhibitory effect of SAMHD1 on production of HBV DNA / 中国免疫学杂志

Objective:To study the expression levels of SAMHD1 in different liver cancer cells and the inhibition of HBV DNA production by SAMHD1. Methods:SAMHD1 mRNA and protein levels in Jurkat,THP-1,HepG2,HepG2. 2. 15 and Huh7 were detected by Real-time quantitative PCR and Western blot. SAMHD1 expression in HepG2. 2. 15 was increased by transfecting SAMHD1 over ex-pression vectors or was inhibited by transfecting SAMHD1 specific shRNA, and then HBV DNA levels were detected. The effect of different dNTP concentration on HBV DNA production was detected also. Results:SAMHD1 were highly expressed in liver cancer cells. Decreased SAMHD1 expression could enhance HBV DNA production by 2. 3-2. 8 times,and increased SAMHD1 expression could inhibit HBV DNA levels by 3 times(P<0. 01). Increased dNTP concentration could enhance HBV DNA production and counteract the inhibitory effect of SAMHD1. Conclusion:SAMHD1 can inhibit the production of HBV DNA in liver cancer cells by decreasing dNTP concentration.

Mechanism of Roscovitine in inhibiting HBV replication / 中华微生物学和免疫学杂志

Objective To investigate the mechanism of Roscovitine, an inhibitor of cyclin-dependent kinase (CDK), in inhibiting HBV replication.Methods Recombiant expression plasmids of SAMHD1 (sterile alpha motif and histidine/aspartic acid domain-containing protein 1) mutants that were defective in dNTPase (deoxynucleoside triphosphate triphosphohydrolase) activity and phosphorylation at the threonine (T) 592 residue were constructed.Huh7.0 cells were respectively co-transfected with different SAMHD1 mutants in combiantion with HBV replication plasmid to analyze whether the retroviral restriction ability of SAMHD1 was regulated by phosphorylation.The cytotoxicity of Roscovitine to Huh7 cells was evaluated by MTT assay.HBV core-associated DNA levels and phosphorylation of SAMHD1 in transfected Huh7.0 cells which were treated with different concentrations of Roscovitine were measured by Southern blot and Western blot assays.Results The SAMHD1 mutant that was defective in the dNTPase active site of D207N lost its ability to restrict HBV replication.Dephosphorylation of SAMHD1 at T592 enhanced its restriction on HBV.The median toxic concentration (TC50) of Roscovitine was 11.20 μmol/L.Both the HBV core-associated DNA levels and the phosphorylation of SAMHD1 were down-regulated by Roscovitine.Conclusion The anti-HBV function of SAMHD1 in dividing cells is regulated by phosphorylation.Roscovitine can inhibit the replication of HBV through reducing the phosphorylation of SAMHD1.

IFN-αcould induce the expression of SAMHD1 by down-regulating miR-181a / 中华微生物学和免疫学杂志

Objective To investigate whether the expression of sterile alpha motif and histidine/aspartic acid domain containing protein 1 ( SAMHD1 ) could be induced by IFN-α and mediated by microRNA-181a (miR-181a). Methods THP-1 and Jurkat cells were treated with different doses of IFN-α(200 IU/ml and 1 000 IU/ml) for 24 h. The expression of miR-181a and SAMHD1 at mRNA level were de-tected by quantitative PCR. Western blot assay was performed to measure the expression of SAMHD1 at pro-tein level. THP-1 and Jurkat cells were transfected with p-181a, an over-expression vector of miR-181a, and then treated with 200 IU/ml of IFN-α. Changes in the expression of SAMHD1 in those cells were analyzed. Results IFN-α significantly enhanced the expression of SAMHD1 at mRNA and protein levels, but inhibi-ted the expression of miR-181a, especially in Jurkat cells. The expression of SAMHD induced by IFN-αwas inhibited in cells over-expressing miR-181a. Conclusion This study suggests that IFN-α could induce the expression of SAMHD1 by down-regulating the level of miR-181a.

The updated research progress of SAMHD1 on the restriction of HIV/SIV replication / 中国比较医学杂志

The host protein SAMHD1 has been identified as the first mammalian deoxynucleoside triphosphate triphosphohydrolase (dNTPase), which blocks the infection of HIV-1 in non-cycling immune cells.SAMHD1 protein is highly expressed in human myeloid-lineage cells and resting CD4+T lymphocytes, which restricts HIV-1 replication by hydrolyzing the cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA ( cDNA) synthesis. Recent studies have revealed that SAMHD 1 plays an important role in virus whole life by promoting HIV -1 genome recombination, degenerating viral genome RNA and restricting virus transmission between cells .In this review, these progress on SAMHD1 research are summarized and the mechanisms by which SAMHD 1 mediates retroviral restriction are analyzed .