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Human and mouse SAMHD1 proteins block human immunodeficiency virus type 1 (HIV-1) infection in noncycling human monocytic cells by reducing the intracellular deoxynucleoside triphosphate (dNTP) concentrations. Phosphorylation of human SAMHD1 at threonine 592 (T592) by cyclin-dependent kinase 1 (CDK1) and cyclin A2 impairs its HIV-1 restriction activity, but not the dNTP hydrolase activity, suggesting that dNTP depletion is not the sole mechanism of SAMHD1-mediated HIV-1 restriction. Using coimmunoprecipitation and mass spectrometry, we identified and validated two additional host proteins interacting with human SAMHD1, namely, cyclin-dependent kinase 2 (CDK2) and S-phase kinase-associated protein 2 (SKP2). We observed that mouse SAMHD1 specifically interacted with cyclin A2, cyclin B1, CDK1, and CDK2. Given the role of these SAMHD1-interacting proteins in cell cycle progression, we investigated the regulation of these host proteins by monocyte differentiation and activation of CD4 ؉ T cells and examined their effect on the phosphorylation of human SAMHD1 at T592. Our results indicate that primary monocyte differentiation and CD4؉ T-cell activation regulate the expression of these SAMHD1-interacting proteins. Furthermore, our results suggest that, in addition to CDK1 and cyclin A2, CDK2 phosphorylates T592 of human SAMHD1 and thereby regulates its HIV-1 restriction function. IMPORTANCE SAMHD1 is the first dNTP triphosphohydrolase found in mammalian cells. Human and mouse SAMHD1 proteins block HIV-1 infection in noncycling cells. Previous studies suggested that phosphorylation of human SAMHD1 at threonine 592 by CDK1and cyclin A2 negatively regulates its HIV-1 restriction activity. However, it is unclear whether human SAMHD1 interacts with other host proteins in the cyclin A2 and CDK1 complex and whether mouse SAMHD1 shares similar cellular interacting partners. Here, we identify five cell cycle-related host proteins that interact with human and mouse SAMHD1, including three previously unknown cellular proteins (CDK2, cyclin B1, and SKP2). Our results demonstrate that several SAMHD1-interacting cellular proteins regulate phosphorylation of SAMHD1 and play an important role in HIV-1 restriction function. Our findings help define the role of these cellular interacting partners of SAMHD1 that regulate its HIV-1 restriction function.

The interferon-induced transmembrane proteins (IFITMs) restrict infection by numerous viruses, yet the importance and regulation of individual isoforms remains unclear. Here, we report that murine IFITM1 (mIFITM1) is palmitoylated on one nonconserved cysteine and three conserved cysteines that are required for anti-influenza A virus activity. Additionally, palmitoylation of mIFITM1 regulates protein stability by preventing proteasomal degradation, and modification of the nonconserved cysteine at the mIFITM1 C terminus supports an intramembrane topology with mechanistic implications.T he interferon-induced transmembrane proteins (IFITMs) have been described to inhibit infection by a wide range of viruses, including important human pathogens such as influenza virus, West Nile virus, severe acute respiratory syndrome (SARS) coronavirus, human immunodeficiency virus, and dengue virus (1-6). The viruses that are restricted share the common ability to enter cells through endocytosis, and the restriction is indeed dependent upon the glycoproteins used for cellular entry (1, 2). Inhibition by IFITM3 occurs prior to influenza virus fusion with the endosome (7), which is a primary site of IFITM3 localization (2,7,8). IFITM3 appears to promote acidification or merging of endosomes with lysosomes, thereby exposing viral particles to a degradative environment (7-9). Consistent with this model, IFITM3 has recently been shown to reduce membrane fluidity and alter the curvature of membranes in such a way that virus hemifusion from within the endosome is inhibited while the fusion of opposing membranes possessing IFITMs may be enhanced (10).The mechanism by which IFITMs change membrane curvature and the endolysosomal compartment is unknown. Addressing this question, we have recently proposed that the predicted dual transmembrane topology for IFITM3 is incorrect and that the active protein instead contains intramembrane domains that do not fully span the lipid bilayer ( Fig. 1A and B) (8). Partial insertion of intramembrane domains into the membrane bilayer is well understood to promote curvature by virtue of the bilayer couple effect (Fig. 1B) (11). Evidence for this topology emerges largely from analysis of posttranslational modifications of IFITM3. Mass spectrometry and mutational analysis identified ubiquitination of lysine 24 (K24) on the IFITM3 N terminus (8). If the predicted topology is adopted, this residue would reside in the endoplasmic reticulum (ER) or endosomal lumen. However, known ubiquitin ligases are cytoplasmic, suggesting that the IFITM3 N terminus is localized in the cytoplasm. This is also supported by the discovery that phosphorylation of IFITM3 on tyrosine 20 (Y20) by the cytoplasmic kinase Fyn is required for IFITM3 antiviral activity (12). We further tested this alternative topology hypothesis by demonstrating that neither the N nor the C terminus nor the central loop domain of IFITM3 is exposed to the ER lumen glycosylation machinery but an antivirally active IFITM3 mutant engineered to possess myristoy...