Figure 1.
SAMHD1 expression in various mouse tissues.
(A) Recombinant mouse SAMHD1 (rSAMHD1) and lysates from parental U937 or U937 expressing human or mouse SAMHD1 were analyzed on an immunoblot probed with rabbit anti-mouse SAMHD1 ISF2 antiserum or with anti-human SAMHD1 antibody. (B) SAMHD1 in lysates of mouse tissues was analyzed on an immunoblot probed with rabbit anti-mouse SAMHD1 antiserum and anti-GAPDH as a loading control. (C) 1000, 200, 40, 8 and 1.6 ng of recombinant mouse SAMHD1 protein and 15 µg of protein from cell lysates from mouse T cells, Con-A-activated T cells, splenocytes and BMDM were analyzed on an immunoblot probed with rabbit anti-SAMHD1 antiserum and anti-GAPDH as a loading control. (D) RNA was extracted from homogenized mouse tissues, and SAMHD1 expression was determined in triplicate as the ratio of SAMHD1 to GAPDH by qRT-PCR using primers recognizing both SAMHD1 isoforms. (E) ISF1 and ISF2 transcript levels were determined in triplicate as their ratio to GAPDH by qRT-PCR using isoform-specific primers. The data are representative of two experiments in A-C. Measurements were in triplicate in D and E and the error bars indicate the standard deviation of the mean.
Figure 2.
SAMHD1 in primary mouse cells is catalytically active.
(A) Mouse T cells, B cells, Con A-activated T cells and LPS-activated B cells from splenocytes, BMDM, RAW264.7 cells and splenocytes were lysed, and SAMHD1 and GAPDH loading control were detected on an immunoblot. (B) SAMHD1 was immunoprecipitated from 0.1 mg or 1.0 mg cell lysates and incubated with [α-32P]-dATP for 240 minutes. The products were visualized by TLC and autoradiography (top panel). Controls included a reaction with buffer alone (buffer), BMDM lysate immunoprecipitated with anti-GST antiserum and a calf intestinal phosphate (CIP)-treated reaction. 10% of the input protein was analyzed on an immunoblot (bottom panel). (C) The catalytic activity of SAMHD1 immunoprecipitated from 70 µg cell lysates was quantified. Measurements were made in duplicate and the error bars indicate the standard deviation of the mean.
Figure 3.
shRNA knock-down of SAMHD1 in the RAW264.7 cell-line enhances retroviral infection.
(A) Cell lysates from RAW264.7 expressing control shRNA or anti-SAMHD1 shRNA were analyzed by immunoblot using rabbit anti-SAMHD1 antiserum with GAPDH detected as a loading control. (B) SAMHD1 knock-down RAW264.7 cells and control cells were infected with 2 µl (7.2×105 cps) and 8 µl (28.8×105 cps) NL-Luc reporter virus and after 3 days, luciferase activity was measured. (C, D) RAW264.7 cells were infected at MOI = 4 with HIV-1-CMV-GFP reporter virus and analyzed 3 days later by flow cytometry. The average of 3 independent experiments is shown in (C). One representative flow cytometry plot for each condition is shown in (D). Cells infected for 6 hours served as a control for baseline fluorescence. (E, F) RAW264.7 cells were infected with MLV-GFP at MOI = 0.1 and 0.4. Three days later, the cells were analyzed by flow cytometry. The average of 3 independent experiments is shown in (E). One representative flow cytometry plot for each condition is shown in (F). (G) The dNTP pool of SAMHD1 knock-down and control RAW264.7 cells was quantified by single-nucleotide extension assay. (H) The growth of SAMHD1 knock-down and control RAW264.7 cells was measured by counting cell numbers over 4 days. SAMHD1 shRNA3 was used in (B-F). Measurements were in triplicate with error bars to indicate the standard deviation of the mean. Statistical significance was calculated using the student’s t-test.
Figure 4.
Knock-down of SAMHD1 in BMDM increases HIV-1 infection.
(A) The time-line shows the scheme for determining the effect of SAMHD1 knock-down on HIV-1 infection of primary BMDM. Bone marrow cells were differentiated to BMDM in M-CSF and then transfected with siRNA. The cells were then infected with luciferase or GFP reporter virus and the infection was quantified after 4 days. (B) BMDM were transfected with individual SAMHD1 siRNAs (siRNA1, 2 and 4), a pool of 4 SAMHD1 siRNAs (siPool) or control siRNA (siControl). SAMHD1 was then detected on an immunoblot probed with rabbit anti-SAMHD1 antiserum. (C) BMDM were transfected with the pool of four siRNAs targeting mouse SAMHD1 (siPool) or control siRNA (siControl) and then infected with NL-Luc reporter virus 4 days later. After 4 more days, the cells were lysed and luciferase activity was measured. (D, E) BMDM were transfected with anti-SAMHD1 siRNA or control siRNA and, after 4 days, infected with HIV-1-CMV-GFP reporter virus. The cells were analyzed 4 days post-infection by flow cytometry. The result of 3 independent experiments is shown (D). Representative flow cytometry data from a single experiment is shown (E). (F) BMDM were transfected with SAMHD1 siRNA pool or control siRNA. After 6 days, extracts were prepared and the dATP concentration was quantified. These experiments were in triplicate with error bars to indicate the standard deviation of the mean.
Figure 5.
Mouse SAMHD1 is inducible by type-I and type-II IFN.
(A) BMDM and RAW264.7 cells were treated for 24 hours with increasing concentrations of IFNβ. SAMHD1 was then detected on an immunoblot probed with anti-SAMHD1 antiserum, and GAPDH was detected as a loading control. The bands were quantified, and the quantification is graphed in the right panel. (B) BMDM and RAW264.7 cells were treated for 24 hours with increasing concentrations of IFNγ. SAMHD1 was detected and quantified as in (A). The data are representative of 3 independent experiments.
Figure 6.
Type-I IFN is regulated by SAMHD1 in RAW264.7 cells.
(A) Total RNA was extracted from SAMHD1 knock-down and control RAW264.7 cells. IFNβ, MX-1, IFIT-1, Trim5, IP-10 and TNFα transcript levels were quantified in triplicate by qRT-PCR, normalized to GAPDH, and the ratio (SAMHD1 knock-down cells to control cells) is shown. (B) Supernatant was collected from SAMHD1 knock-down and control RAW264.7 cells and IFNβ was quantified. Measurements were in triplicate and error bars indicate the standard deviation.