Restricted HIV replication kinetics correlates with SAMHD1 expression. (A) 1 × 106 cells/ml astrocytes and microglia were infected with HIV-1BAL (100 ng p24) for overnight, washed and replenished with fresh medium. Virus replication kinetics is quantitated in cell culture supernatant over a 20-day time course, by the production of p24 antigen. (B) Cells were infected with the HIV-luciferase pseudovirus (20 ng) for 3 days, harvested, washed with PBS and lysed. Luciferase concentration in whole cell lysates was determined. (C) RT-PCR based quantification of SAMHD1 mRNA levels. Total cellular RNA was extracted from microglia and astrocytes (infected with HIV-1BAL) and amplified by specific primer for SAMHD1 gene expression. All data was normalized to GAPDH. Data shown represents fold change in mRNA levels. (D) SAMHD1 protein expression was detected by immunoblotting with SAMHD1-specific antibodies in corresponding cell lysates. Equal amounts of total protein (30 μg) of the lysates from both cells were loaded. GAPDH was used as a loading control. (E) Intracellular SAMHD1 expression of microglia and astrocytes. Cells (1 × 106) were immunostained by FITC-conjugated SAMHD1 monoclonal Ab and analyzed by flow cytometry. Percentage of cells expressing SAMHD1 is indicated. (F) Cell culture supernatant from astrocytes and microglia after HIV infection was assessed for viral reverse transcriptase activity over 20 days by colorimetry. (G) Total cellular RNA was extracted from the corresponding cells following HIV infection (over a 20-day time course) and SAMHD1 mRNA levels quantitated by RT-PCR. (H) SAMHD1 protein expression was detected by immunoblotting with SAMHD1-specific antibodies in corresponding cell lysates at 3, 5, 7, 10, and 15 days following HIV infection. Results shown are mean ± SEM of three independent experiments for A, B, C, F, and G. P value ≤0.05 were considered to be significant. SAMHD1, sterile alpha motif and histidine/aspartic acid domain-containing protein 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT, reverse transcriptase; RLU, relative light units.