Figure 2.
SAMHD1 directly degrades HIV-1 RNA in human monocytic cells. (a) The total cellular RNA was extracted from SAMHD1-expressing, HIV-1-GFP-infected U937 cells. The HIV-1 RNA content was quantified by qRT-PCR using HIV-1 gag-specific primers. The data were normalized to an internal β-actin. (b) Mock- and SAMHD1WT-expressing U937 cells were infected with HIV-1-GFP or HIV-1D443N-GFP. The HIV-1 RNA content was quantified by qRT-PCR using HIV-1 gfp-specific primers. (c) Relative HIV-1 genome abundance in reads per kilobase per million mapped reads (RPKM) calculated from RNA-Seq data. (d) Coverage of the RNA-Seq data across the HIV-1 genome in U937 cells expressing SAMHD1WT, SAMHD1D207N and SAMHD1D137N after HIV-1-GFP infection. Data are representative of two independent experiments (c,d). (e) shRNA-mediated silencing of SAMHD1 (left) or Vpx-mediated degradation of SAMHD1 (right) in THP-1 cells increases HIV-1 RNA stability. THP-1 cells expressing shRNAs, Vpx or negative control were treated with PMA for 24 h followed by infection with HIV-1-GFP. The total viral RNA was quantified by qRT-PCR at the indicated times post-infection. Knockdown efficiency was determined by immunoblot analysis. (f) Enrichment of HIV-1 genomic RNA in the SAMHD1 immunoprecipitates. U937 cells expressing HA-tagged SAMHD1 variants were infected for 90 min with HIV-1-GFP. The purified RNAs in the anti-HA immunoprecipitates were quantified by qRT-PCR using HIV-1 gag-specific primers. In a, b, e, and f, data are presented as the mean ± s.d. of triplicate independent experiments. (*P < 0.05 and **P < 0.001 versus Mock or shControl, two-tailed Student's t test).