Identification of Cellular Proteins Interacting with the Retroviral Restriction Factor SAMHD1

Abstract

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 dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

“…In order to further confirm our data we tested whether cyclin E, similar to cyclin A2 [14,16], interacts directly with SAMHD1. We prepared full-length or deletion derivatives of human SAMHD1 fused at the N-terminus with GST (upper panel of Figure 3(a)) and tested their ability to pull down in vitro translated S 35 -labeled cyclins E and A2.…”

“…Earlier reports identified SAMHD1 as an interacting partner of Skp2 in proliferating cells [16]. Skp2 is a component of the SCF ubiquitin ligase that recognizes phosphorylated proteins and regulates the proteolytic events driving cells through the G1/S transition.…”

“…SAMHD1 is phosphorylated at threonine 592 (T592) by the cell-cycle regulated kinases CDK2/1 [14–16]. Phosphorylated T592 is believed to have a regulatory function but how it relates to SAMHD1 activity and/or protein stability is still questioned.…”

SAMHD1 Specifically Affects the Antiviral Potency of Thymidine Analog HIV Reverse Transcriptase Inhibitors

“…Although differences in expression between cell lines and primary tissues exist, SAMHD1 expression is similar between HIV-1-resistant and -susceptible cells (6,17). Posttranscriptional control by phosphorylation has been proposed as a plausible hypothesis underlying differences in SAMHD1 function between resistant and susceptible cells, although it has not been experimentally proved yet (24)(25)(26). Here, we evaluated SAMHD1 activity in SAMHD1-negative or low-expressing transformed cell lines, as well as in primary cells, demonstrating that SAMHD1 activity is maintained in the absence of viral restriction and is specifically influencing thymidine NRTI analog antiviral efficacy.…”

“…Similarly, SAMHD1 antiretroviral activity is modulated by cell cycle-dependent posttranslational modifications. Phosphorylation of SAMHD1 by cyclin-dependent kinases (CDK), whose activation is tightly controlled depending on the moment of the cell cycle (36), have been demonstrated as the regulatory mechanism of SAMHD1-mediated viral restriction in cycling cells (24)(25)(26). Our results do not point to a SAMHD1-dependent regulation of TK1 function, as similar results were obtained in different cell types, i.e., cycling cells (immortalized cell lines and activated lymphocytes) or differentiated cells (macrophages), expressing different levels of SAMHD1 (Fig.…”