Figure 7: Tetramer formation of SAMHD1 is required for dNTPase activity and HIV-1 regulation.
(a–f) Mutations of residues involved in dGTP binding in the allosteric site of SAMHD1 affect tetramer formation. Purified recombinant SAMHD1c′ and SAMHD1c′ mutant proteins were incubated with 50 μM dGTP and separated by size-exclusion chromatography. The elution profiles were monitored by ultraviolet absorbance at A280 nm and compared with known molecular size markers. (g) Mutations of residues involved in dGTP binding in the allosteric site of SAMHD1c′ affect dNTPase activity. Purified recombinant SAMHD1c′ and SAMHD1c′ mutant proteins were analysed for dNTPase activity as described in Methods, and the activity of SAMHD1c′ was set to 100%. The s.e. from triplicate samples (n=3) is shown. (h) Tetramer formation is required for SAMHD1-mediated restriction of HIV-1. U937 cells were transduced with pLVX puro-based SAMHD1 expression vectors. Transduced U937 cells were differentiated into macrophages by PMA (phorbol 12-myristate 13-acetate) treatment and infected with HIV-1 EGFP viruses. GFP-positive cells were analysed 2 days after infection by flow cytometry. The experiment was repeated for three times (n=3). The error bars indicated the s.d. of three replicates. Student’s t-test was performed with Microsoft Excel to generate P-values.
