Figure 4.

Test for bSAMHD1 degradation by BIV and eSAMHD1 degradation by EIAV. A and B, TLC-based (A) and HPLC-based (B) dNTPase activity assays of bSAMHD1 protein. Both TLC- and HPLC-based dNTPase assay were conducted with purified bSAMHD1 protein. These assays were performed in triplicates as described for fSAMHD1 in Fig. 1. [α-³²P]dTTP and dGTP were used as SAMHD1 substrates in TLC- and HPLC-based assays, respectively. PPPi, triphosphate product. The HPLC data show the dGTP hydrolysis and dG product by WT bSAMHD1 (blue line) and HD mutant (red line). dCMP is the internal loading control. C, test for degradation of bSAMHD1 by BIV. Human 293T cells were co-transfected with pLVX-IRES-mCherry co-expressing HA-tagged bSAMHD1 and mCherry protein (pBovine SAMHD1) (0.1 μg) and either a full-length molecular clone of BIV (pBIV 127) (2 μg), or pLVX-IRES-mCherry expressing on mCherry protein (pLVX-IRES-mCherry) (2 μg). The bSAMHD1 protein levels were determined in Western blotting with HA antibody. GAPDH was used as a loading control. D, test for degradation of eSAMHD1 by EIAV. Human 293T cells were co-transfected with pLVX-IRES-mCherry co-expressing HA-tagged eSAMHD1 and mCherry protein (pEquine SAMHD1) (0.1 μg) and either EIAV vector packaging plasmid (pEIAV packaging) (2 μg) or GFP-expressing EIAV vector transfer plasmid (pEIAV-GFP transfer) (2 μg). The eSAMHD1 protein levels were determined by Western blotting with HA antibody. GAPDH was used as a loading control. The Western blotting data presented in this figure are representative data from more than three independent transfections, and mean relative SAMHD1 levels shown were calculated by densitometry analysis. The calculated mean ± S.D. values are (C) 2.69 ± 0.40 and (D) 1.29 ± 0.27.