Identification of Cellular Proteins Interacting with the Retroviral Restriction Factor SAMHD1
- Corine St. Gelaisa,
- Suresh de Silvaa,
- Jocelyn C. Hachb,
- Tommy E. Whitec,
- Felipe Diaz-Grifferoc,
- Jacob S. Yountb and
- Li Wua,b
- aCenter for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, USA
- bDepartment of Microbial Infection and Immunity, The Ohio State University, Columbus, Ohio, USA
- cDepartment of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, New York, USA
- R. W. Doms, Editor
-
FIG 1Identification of cellular proteins interacting with human and mouse SAMHD1. (A) HEK293T cells were transfected with constructs expressing HA-tagged human or mouse SAMHD1 (HA-hSAMHD1 and HA-mSAMHD1, respectively) or HA-tagged mouse CD9 (HA-mCD9) as a negative control. Cell lysates were harvested the following day and coimmunoprecipitated using anti-HA-coated agarose. Interacting proteins were eluted using an HA peptide, and samples were analyzed by SDS-PAGE and Coomassie blue staining. Gel slices were excised and processed for mass spectrometry analysis. (B) HEK293T cells were transfected as described for panel A, and the empty vector was used as a negative control. Eluted lysates were used to validate mass spectrometry results by performing coimmunoprecipitation using anti-HA and immunoblotting for the identified cellular proteins using antibodies specific to human cyclin A2, CDK2, SKP2, cyclin B1, and CDK1 (the order is based on the peptide abundance identified by mass spectrometry [Table 1]).
-
FIG 2Regulation of SAMHD1-interacting proteins by monocyte differentiation and T-cell activation. (A) Regulation of the expression of SAMHD1 and its interacting proteins in monocytic cell lines by PMA treatment for 36 h. Cells were cultured and mock or PMA treated. Cell lysates (20 μg) were used for protein detection by immunoblotting, and GAPDH was used as a loading control. (B) Regulation of the expression of SAMHD1 and its interacting proteins in primary monocytes and monocyte-derived dendritic cells (DCs). DCs were differentiated from autologous monocytes, and cell lysates were analyzed by immunoblotting. Data shown represent one of two donors analyzed. (C) Regulation of the expression of SAMHD1 and its interacting proteins by activation of CD4+ T cells and PBMCs by either anti-CD3/CD28 or PHA treatment for 72 h. Cell lysates (10 μg) were analyzed by immunoblotting. Data shown represent one of two donors analyzed. (D) Activation of primary resting CD4+ T cells and PBMCs. Primary CD4+ T cells and PBMCs were isolated from healthy donors and activated by treatment with PHA or anti-CD3/CD28 in the presence of IL-2. Immunostaining of the T-cell activation markers CD25 was analyzed by flow cytometry.
-
FIG 3Inhibitors of CDK1 and CDK2 block T592 phosphorylation of SAMHD1 and reduce HIV-1 infection in THP-1 cells. (A) HEK293T cells were pretreated with either DMSO (vehicle control) or inhibitors to CDK1, CDK2, and SKP2 at the assigned concentrations and transiently transfected to overexpress SAMHD1. The effect of each individual inhibitor on the phosphorylation of SAMHD1 at T592 was assessed at 30 h post-inhibitor treatment by immunoblotting. (B) THP-1 cells were treated with either DMSO or inhibitors to CDK1, CDK2, and SKP2 at the concentrations indicated for 30 h. Endogenous total or T592-phosphorylated SAMHD1 protein levels were determined by immunoblotting using specific antibodies. GAPDH was used as a loading control. Relative phospho-T592 levels represent T592-phosphorylated hSAMHD1 normalized to GAPDH. Relative hSAMHD1 levels represent hSAMHD1 normalized to GAPDH. Protein bands were quantified and normalized to the respective DMSO-treated controls, which were set to 1. The data presented are representative of 3 independent experiments. (C) Inhibitors of CDK1 and CDK2 reduce HIV-1 infection in THP-1 cells. Cells were pretreated with either DMSO control or inhibitors of CDK1 or CDK2 and then cultured in the presence of the inhibitors for 24 h at the concentrations indicated. At 24 h post-inhibitor treatment, cells were challenged with single-cycle HIV-Luc/VSV-G at an MOI of 0.5 for 2 h; cells were then washed and cultured in fresh media containing inhibitor for 24 h, and lysates were harvested for luciferase assay. All infections were calculated relative to the DMSO controls, which were set as 1. Data presented are representative of 2 independent experiments, and error bars show the standard deviations of triplicate samples. *, P < 0.05 (compared to the DMSO controls without inhibitors).
-
FIG 4Expression of dominant negative (DN) mutants of CDK1 and CDK2 reduces T592 phosphorylation of hSAMHD1. (A and B) Overexpression of hSAMHD1 and wild-type (WT) or DN mutants of HA-tagged CDK1 or CDK2 (A) or FLAG-tagged SKP2 (B) in HEK293T cells. Empty vector was used as a negative control. A 2-fold increase of plasmid DNA-expressing DN mutants was applied in the cotransfection as indicated. At 24 h posttransfection, cell lysates were harvested for immunoblotting to determine overexpression of transfected DNA using either HA- or FLAG-specific antibodies. The effect on phosphorylated SAMHD1 levels was determined using a phospho-T592-specific antibody. GAPDH was used as a loading control. Relative phospho-T592 levels represent T592-phosphorylated hSAMHD1 normalized to GAPDH. Protein bands were quantified and normalized to the respective WT controls, which were set to 1. The data presented are representative of 3 independent experiments.
-
FIG 5Knockdown of CDK1, CDK2, and cyclin A2 in HEK293T cells reduces T592 phosphorylation of SAMHD1. siRNA-mediated knockdown of cyclin A2, CDK1, or both (double) (A) and cyclin A2, CDK2, or both (double) (B) in HEK293T cells was performed using a two-round transfection protocol. At 24 h after the second round of siRNA transfection, cell lysates were harvested for immunoblotting to determine efficient knockdown of target proteins. GAPDH was used as a loading control. Relative cyclin A2 and CDK1 levels shown represent GAPDH-normalized densitometry compared to scramble siRNA control. Cells that had undergone a single round of siRNA transfection were then dually transfected with a second round of siRNA as well as a plasmid to overexpress wild-type hSAMHD1. Cell lysates were harvested at 24 h after the second round of siRNA transfection, and levels of phosphorylated (Phospho-T592) and total hSAMHD1 proteins were determined by immunoblotting. Relative phosphorylated T592 levels represent phosphorylated hSAMHD1 normalized to total hSAMHD1, compared to scramble control, which was set as 1. Membranes to be immunoblotted were cut and probed for each antibody accordingly as shown (cyclin A2, CDK1, CDK2, GAPDH, T592 phosphorylated hSAMHD1, and total hSAMHD1). The data presented are representative of 3 independent experiments.
-
FIG 6Unphosphorylated form of hSAMHD1 at T592 restricts HIV-1 infection in differentiated U937 cells, but not in cycling HEK293T cells. (A and B) HEK293T cells were transfected with vector control or plasmids expressing wild-type (WT) hSAMHD1 or a T592A mutant. (A) At 24 h posttransfection, lysates were harvested for immunoblotting to confirm overexpression of total hSAMHD1 and T592 phosphorylated hSAMHD1 (Phospho-T592). (B) High efficiency of transfection in HEK293T cells. Cells transfected with a GFP-expressing vector were harvested and analyzed by flow cytometry at 24 h posttransfection to determine GFP expression (95% cells were positive for GFP). (C) Transfected HEK293T cells were infected with single-cycle HIV-Luc/VSV-G at an MOI of 0.5. At 24 h postinfection, cell lysates were harvested for a luciferase assay to determine HIV-1 infection. The fold change of HIV-1 infection is shown. The data presented are representative of 2 independent experiments. (D) U937 cells stably expressing hSAMHD1 (WT) and hSAMHD1 (T592A) mutant were treated with PMA for 24 h and subjected to immunoblotting to assess the expression level of WT and T592A mutant proteins. (E) A parallel set of samples that were PMA treated in a similar manner were infected with HIV-Luc/VSV-G at an MOI of 2, and infection levels were assessed at 24 h by measuring the luciferase activity. The data presented are representative of 3 independent experiments, and error bars show the standard deviations of triplicate samples. *, P < 0.001 (compared to the vector control).
- Copyright © 2014, American Society for Microbiology. All Rights Reserved.
