Nature 474:658C661 [PMC free article] [PubMed] [Google Scholar] 4. these residues to alanine to prevent phosphorylation or to glutamic acid to mimic phosphorylation experienced no effect on the nuclear localization of SAMHD1 or its level of sensitivity to Vpx-mediated degradation. Furthermore, neither alanine nor glutamic acid substitutions experienced a significant effect on SAMHD1 dNTPase activity in an assay. Interestingly, however, we found that a T592E mutation, mimicking constitutive UNC0379 phosphorylation at a main phosphorylation site, seriously affected the ability of SAMHD1 to restrict HIV-1 inside a U937 cell-based restriction assay. In contrast, a T592A mutant was still capable of restricting HIV-1. These results indicate that SAMHD1 phosphorylation may be a negative regulator of SAMHD1 restriction activity. This conclusion is definitely supported by our finding that SAMHD1 is definitely hyperphosphorylated in monocytoid THP-1 cells under nonrestrictive conditions. Intro Lentiviruses, such as HIV and SIV, encode several accessory proteins that function to counteract sponsor cell restriction factors (examined in research 1). Sterile alpha motif UNC0379 and HD website protein 1 (SAMHD1) is definitely a recently recognized host cell element targeted from the HIV-2 and Rabbit Polyclonal to AMPKalpha (phospho-Thr172) SIVsm encoded Vpx protein to allow replication of these viruses in myeloid cells (2C4). Interestingly, while HIV-1 does not possess a Vpx protein, Vpx also enhances illness of myeloid and dendritic cells, as well as resting CD4+ T cells by this disease (5C10). In vulnerable cell types, SAMHD1 offers been shown to restrict illness of these lentiviruses in the reverse transcription step, and Vpx counteracts this restriction by binding to and causing the proteasomal degradation of SAMHD1 via connection having a Cul4/DDB1/DCAF1 ubiquitin-ligase complex (2, 3, 11). Similarly, without Vpx, the same enhancement of HIV-1 illness in these cell types can consequently be achieved UNC0379 from the knockdown of SAMHD1 (2C4, 9). SAMHD1 consists of an N-terminal SAM website and a C-terminal HD website and mutations in SAMHD1 have been associated with Aicardi-Goutieres Syndrome (AGS) (12). This syndrome is definitely associated with improved production of interferon alpha and therefore mimics congenital infections (13). Mutations in two additional proteins (TREX1 and RNaseH2) have also been associated with AGS, and it has therefore been suggested that all three of these proteins may be involved in regulating the innate immune response (14). While SAMHD1 has recently been shown to possess nucleic acid binding properties (15C18) and in one study was also reported to have exonuclease activity (17), its main catalytic activity explained to date is definitely its dGTP-dependent deoxynucleoside triphosphohydrolase (dNTPase) activity that allows it to degrade cellular deoxynucleoside triphosphates (dNTPs) (19, 20). In this way, SAMHD1 is definitely thought to restrict HIV-1 illness by reducing the levels of cellular dNTP swimming pools to below that required for reverse transcription (19C22). Interestingly, while SAMHD1 offers been shown to reduce HIV-1 illness of nondividing cell types such as UNC0379 MDMs, dendritic cells, resting CD4 T cells as well as phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 and U937 cells (the second option requiring exogenous manifestation of SAMHD1) (2C4, 9, 10, 23), SAMHD1 restriction does not purely correlate with its manifestation. Indeed, fully HIV-1 permissive cells, such as triggered CD4+ T cells or undifferentiated THP-1 cells, also communicate high amounts of the SAMHD1 protein (3, 9). Whether additional mechanisms exist to keep the dNTP levels high in these dividing cells and/or whether SAMHD1 function might be controlled at the level of posttranslational modifications or connection with cell specific cofactors remains to be determined. Here, we statement that SAMHD1 can be phosphorylated at several sites, and this suggests a mechanism to regulate its cellular function. We display that phosphorylation of SAMHD1 at any of the four recognized positions did not significantly affect protein stability, localization, or level of sensitivity to Vpx-mediated degradation. Mutation of any of the phosphorylation sites also experienced no significant effect on dNTPase catalytic activity of SAMHD1 for 10 min at 4C. Cleared lysates were then incubated for 1.5 h at 4C with antibody (SAM416)-conjugated protein UNC0379 A-Sepharose beads. Beads were washed three times with wash buffer (50 mM Tris [pH 7.5], 300 mM NaCl, 0.1% Triton X-100, 1 mM Na3VO4, 1 mM NaF). Bound proteins were eluted in sample buffer for 10 min at 95C, separated by SDS-PAGE, transferred to a PVDF membrane, and analyzed by immunoblotting with.