The Function Of SAMHD1 For Anti-virus

The dNTP hydrolyses SAM domain and HD domain-containing protein 1 (SAMHDI) functions as a retroviral restriction factor in myeloid dendritic cells, macrophages and resting CD4+T-cells, and by reducing the level of intracellular dNTP, restricts retrovirus replication. Moreover, HIV-2 and some simian immunodeficiency viruses (SIVs) can degrade SAMHDI. The viral accessory protein Vpx induces proteasomal degradation of SAMHDI by promoting binding to CRL4DCAF1 E3 ubiquitin ligase. Degradation of SAMHDI promotes HIV-1 reverse transcription, enhancing infection of human mononuclear cells, dendritic cells and macrophages. We sought to explore the antiviral function of SAMHDI by usingSAMHD1 mutants, and assessing SAMHDI expression in CEMx174 infected with SIVmac239 and rhesus monkeys infected withSHIVSF162P3·PART 1:The research of different SNP sites of SAMHDI antiviral protein function.Large-scale gene screening in rhesus monkey identified five SNPs that are prevalent in the population. Three SNPs,G183S, E184D, V280A, are distributed throughout the HD structure domain, which has phosphoric acid hydrolysis enzyme activity, and S107L is located in the SAM structure, which participates in protein-protein and protein-nucleic acid interactions.Prodictprotein software was used to analyze the impact of the five SNP sites on SAMHD1 secondary and tertiary structure. We found that G183S, E184D, V280A are most likely to affect the structure and function of SAMHDI by converting an alpha helix domain into a beta lamella. This alteration in secondary structure may alter protein binding sites. V280A was located in the opening of the protein activity center grooves, a lid structure that could affect SAMHDI phosphoric acid hydrolysis catalytic activity. The S107L site is reported to located SAMHDI sensitivity site and affect bing virus Vpx protein, which could explain the effect of this site on SAMHDI function.D5E is not located in an important structural domain, and there is no evidence to demonstrate that this SNP altersSAMHD1 activity. However, the N terminal of SAMHDI can interact with HIV, so further experiments will be required to clarify the role of this mutation.In this study, we used an expression vector to transfect TZM-bl cells with mutant SAMHD1. We then measured HIV and SIV infection in these cells to assess the impact of these SNPs onSAMHD1 antiviral function. We found that HIV infection level were not different between cells transfected with D5E, G183S, V280A or wild type SAMHD1, however values in cells transfected withS107L SAMHD1 TCID50 were 10 fold lower, indicating an enhanced antiviral function of S107L SAMHD1. Values in cells transfected withE184D SAMHD1 TCIDsowere 10 higher, indicating a reduced antiviral function of E184D SAMHD1.In contrast, the level of SIVmac239 infection were not different between cells transfected with mutant and wild type SAMHD1, likely due to the SIV encoded Vpx protein. It also suggests that, as a result of the existence of the SIV Vpx protein which can degrade the SAMHD1 protein, it is not too big relations between SIV infected rhesus differences disease progression and rhesus monkeys SAMHD1 protein, and has nothing to do with the different SNP sites SAMHD1 protein.PART 2:The relationship between SAMHD1 expression and SIVmac239 infection of CEMx 174 cells.SAMHD1 acts as restriction factor of HIV infection of myeloid dendritic cells, macrophages and resting CD4+T cells, by reducing the level of intracellular dNTP. However in the dNTP level in activated CD4+T cells is significantly higher than that in restingCD4+T cells, and does not significantly decrease after infection. It was speculate that SAMHD1 does not exert an antiviral effect in activated CD4+T cells. However, in addition to degradation of dNTP,SAMHD1 was recently reported to exert antivural RNase activity. This activity would not necessarily be inhibited in CD4+T cells.In this study, we infected CEMx 174 cells with SIVmac239 and detected the viral load in the culture supernatant and P27 protein of the cells. Four days after infection cellular SAMHD1 mRNA levels were significantly elevated, and positively correlated with viral load. However, after day 4 the level of SAMHD1 protein expression decreased, was undetectable on days 5 and 6, and was negatively correlated with SIV P27 protein level.PART3:Expression of SAMHD1 mRNA is altered in SHIVSF162P3 infected rhesus monkeys.Bianka Mubil and others previously reported that mRNA levels of the antiviral proteinsA3G and A3F are elevated in SIV infected rhesus monkeys during the asymptomatic phase of disease. Whether SAMHD1 expression in SIV/SHIV infected monkeys reflects changes in viral load, or plays an antiviral function in rhesus monkeys remains to be determined.We infected four Chinese rhesus monkeys with SHIVSF162p3, one of which exhibited characteristics of rapid disease progression. We used a relative quantitative method to assess the level of SAMHD1 mRNA in rhesus peripheral blood mononuclear cells (PBMC). In three monkeys, SAMHD1 expression increased as the viral load increased, in addition to the rapid progress of G0822V; Rapid progress of G0822V is the opposite, with consistently high levels of viral load, SAMHD1 mRNA level downward trend of fluctuations. We believe that SAMHD1 mRNA expression may be associated with the disease progression, and high viral loads can downregulateSAMHD1 expression.We also established a method for real-time fluorescence quantification of SAMHD1 mRNA. We found that SAMHD1 mRNA expression fell to baseline levels following the acute phase of infection. Thus we think that although SAMHD1 acts as an antiviral protein, SAMHD1 expression may be reduced either directly by viral infection or by an immune response to viral infection. Additionally SHIVSF162P3infection of mononuclear cells and monocyte-derived macrophages induces apoptosis of these cells, likely reducing SAMHD1 expression as these are the cells in which it is induced.