Contribution of SAM and HD Domains to Retroviral Restriction Mediated by Human SAMHD1
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Virology 436 (2013) 81–90 Contents lists available at SciVerse ScienceDirect Virology journal homepage: www.elsevier.com/locate/yviro Contribution of SAM and HD domains to retroviral restriction mediated by human SAMHD1 Tommy E. White a,1, Alberto Brandariz-Nun˜ez a,1, Jose Carlos Valle-Casuso a, Sarah Amie b, Laura Nguyen b, Baek Kim b, Jurgen Brojatsch a, Felipe Diaz-Griffero a,n a Department of Microbiology and Immunology, Albert Einstein College of Medicine Bronx, NY 10461, USA b Department of Microbiology & Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA article info abstract Article history: The human SAMHD1 protein is a novel retroviral restriction factor expressed in myeloid cells. Previous Received 1 September 2012 work has correlated the deoxynucleotide triphosphohydrolase activity of SAMHD1 with its ability to Returned to author for revisions block HIV-1 and SIVmac infection. SAMHD1 is comprised of the sterile alpha motif (SAM) and histidine– 24 September 2012 aspartic (HD) domains; however the contribution of these domains to retroviral restriction is not Accepted 20 October 2012 understood. Mutagenesis and deletion studies revealed that expression of the sole HD domain of Available online 13 November 2012 SAMHD1 is sufficient to achieve potent restriction of HIV-1 and SIVmac. We demonstrated that the HD Keywords: domain of SAMHD1 is essential for the ability of SAMHD1 to oligomerize by using a biochemical assay. SAMHD1 In agreement with previous observations, we mapped the RNA-binding ability of SAMHD1 to the HD HIV-1 domain. We also demonstrated a direct interaction of SAMHD1 with RNA by using enzymatically-active HD domain purified SAMHD1 protein from insect cells. Interestingly, we showed that double-stranded RNA inhibits Restriction dNTPase the enzymatic activity of SAMHD1 in vitro suggesting the possibility that RNA from a pathogen might RNA binding modulate the enzymatic activity of SAMHD1 in cells. By contrast, we found that the SAM domain is dispensable for retroviral restriction, oligomerization and RNA binding. Finally we tested the ability of SAMHD1 to block the infection of retroviruses other than HIV-1 and SIVmac. These results showed that SAMHD1 blocks infection of HIV-2, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Equine infectious anemia virus (EIAV), N-tropic murine leukemia virus (N-MLV), and B-tropic murine leukemia virus (B-MLV). & 2012 Elsevier Ltd. All rights reserved. Introduction 2008). These experiments suggest that when Vpx is incorporated into viral particles, the virus overcomes a pre-reverse transcrip- Infection of primary macrophages and dendritic cells by tion block in macrophages and dendritic cells. Remarkably, Vpx Simian Immunodeficiency virus (SIVmac) requires the accessory also increases the ability of HIV-1 to infect macrophages and protein Vpx, which is encoded in the SIV genome (Ayinde et al., dendritic cells when Vpx is incorporated into HIV-1 particles or 2010). SIVmac particles without Vpx (SIVDVpx) are unable to infect supplied in trans (Goujon et al., 2008; Sunseri et al., 2011). This primary macrophages. Vpx is essential for both SIV infection of suggests that the block imposed by macrophages to SIVDVpx is primary macrophages and viral pathogenesis in vivo (Belshan similar to the one imposed by macrophages to HIV-1. et al., 2006; Fletcher et al., 1996; Gibbs et al., 1995; Hirsch et al., Recent work identified SAMHD1 as the protein that blocks 1998). Vpx is incorporated into viral particles suggesting that it infection of SIVDVpx, HIV-2DVpx and HIV-1 before reverse tran- might be acting immediately after viral fusion in the target cells scription in macrophages and dendritic cells (Hrecka et al., 2011; (Jin et al., 2001; Kappes et al., 1993; Park and Sodroski, 1995; Laguette et al., 2011). However, SIV, HIV-2 and HIV-1 expressing Selig et al., 1999). Viral reverse transcription is prevented in Vpx overcome the SAMHD1-dependent restriction (Hrecka et al., primary macrophages when cells are infected with either Vpx- 2011; Laguette et al., 2011). Mechanistic studies have suggested that deficient SIVmac or HIV-2 (Bergamaschi et al., 2009; Fujita et al., Vpx induces the proteasomal degradation of SAMHD1 (Berger et al., 2008; Goujon et al., 2007; Kaushik et al., 2009; Srivastava et al., 2011; Hrecka et al., 2011; Laguette et al., 2011). Interestingly, the C- terminal region of SAMHD1 contains a Vpx binding motif, which is important for the ability of Vpx to degrade SAMHD1 (Laguette et al., n Correspondence to: Albert Einstein College of Medicine, 1301 Morris Park, 2012; Lim et al., 2012; Zhang et al., 2012). In addition, findings by Price Center 501, New York, NY 10461, USA. Fax: þ1 718 632 4338. E-mail address: [email protected] (F. Diaz-Griffero). Lim et al. (2012) have suggested that the SAM domain of SAMHD1 is 1 Contributed equally to this work. important for the ability of Vpx to induce SAMHD1 degradation. 0042-6822/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.virol.2012.10.029 82 T.E. White et al. / Virology 436 (2013) 81–90 Fig. 1. Contribution of the different domains of SAMHD1 to restriction of HIV-1. Wild type human SAMHD1 protein is depicted in the top figure. The numbers of the amino acid residues at the boundaries of the SAMHD1 domains are indicated. The HD domain boundaries used for these studies are based on the published structure (Goldstone et al., 2011). SAMHD1 variants are shown, with the broken lines indicating deleted sequences (A). Human monocytic U937 cells stably expressing the indicated mutant and wild type SAMHD1 proteins (B) were challenged with increasing amounts of HIV-1-GFP (C). As control, U937 cells stably transduced with the empty vector LPCX werechallengedwith HIV-1-GFP. (D) Quantification of dATP levels on PMA-treated U937 cells expressing the indicated SAMHD1 variant was performed as described in Materials and methods. SAM, sterile alpha motif; HD, histidine–aspartic. SAMHD1 comprises a sterile alpha motif (SAM) and a histidine– Results aspartic (HD) domain. SAM domains are protein interaction modules that mediate interaction with other SAM domains or non-SAM Contribution of the different domains of SAMHD1 to retroviral domain-containing proteins (Rice et al., 2009). Moreover, SAM restriction domains in other proteins bind a specific sequence of DNA acting as either transcription activators or repressors (Qiao and Bowie, The recently discovered restriction factor SAMHD1 comprises 2005). The HD domain is found in a super family of enzymes with a of a SAM and HD domain. Although the enzymatic activity of the predicted phosphohydrolase activity (Zimmerman et al., 2008). In HD domain is required for the ability of SAMHD1 to block HIV-1 agreement, recent work has demonstrated that SAMHD1 is a dGTP- and SIVmac infection (Goldstone et al., 2011; Lahouassa et al., regulated deoxynucleotide triphosphohydrolase that might be 2012b; Powell et al., 2011), the contribution of the different involved in decreasing the overall cellular levels of triphosphodeox- regions of the SAMHD1 protein to restriction is not understood. ynucleotides (Goldstone et al., 2011; Kim et al., 2012; Lahouassa To understand the contribution of the SAM and HD domain to et al., 2012b; Powell et al., 2011). This evidence agrees with the lentiviral restriction, we generated a series of deletion constructs hypothesis that an overall decrease in the level of dNTPs is to find the minimal requirements for potent restriction (Fig. 1A). responsible for the block imposed to lentiviral infection. The different SAMHD1 variants were stably expressed in the The SAM and HD domains have been extensively studied on human monocytic cell line U937 by using the LPCX vector system other proteins; however, the contribution of the different (Fig. 1B), as previously described (Brandariz-Nunez et al., 2012). domains of SAMHD1 to HIV-1 restriction has not been investi- U937 cells stably expressing SAMHD1 variants were induced to gated. This work explores the contribution of the different differentiation by PMA treatment (Schwende et al., 1996). Differ- domains of SAMHD1 to HIV-1 restriction. By performing a series entiated U937 cells were challenged with increasing amounts of of deletion constructs, we found that the expression of the sole HIV-1 containing the green fluorescent protein as a reporter (HIV- HD domain of SAMHD1 in U937 cells is sufficient to potently 1-GFP) (Fig. 1C and Table 1). Overall these studies showed that block HIV-1 infection. By contrast we showed that the SAM expression of the SAMHD1 variant 112–582, which contains only domain seems to be dispensable for restriction. Using biochemical the HD domain, is sufficient to block HIV-1 infection. In agreement, assays, we demonstrated that the HD domain of SAMHD1 is the SAMHD1 variants D164–319, 1–328 and HD206AA that have either basic requirement for the ability of SAMHD1 to oligomerize and a defective or deleted HD domain completely lost their ability to bind RNA. Furthermore, we explored the ability of SAMHD1 and block HIV-1 infection (Fig. 1CandTable 1). Because deletion of the HD domain to block infection by different retroviruses. the SAM domain in the variant 112–626 did not affect HIV-1 T.E. White et al. / Virology 436 (2013) 81–90 83 Table 1 Phenotypes of SAMHD1 variants. SAMHD1 variant HIV-1 restrictiona Oligomerizationb RNA bindingc Localizationd Cellular dATP levele WT þ þþþ þþþ N Low 1–150 ND ÀÀND ND 151–328 ND ÀÀNþCND 15–626 þþþþþþC Low D45–110 À þþþ þþþ N High D164–319 ÀÀþþþN High 112–626 þ þþþ þþþ C Low 1–328 ÀÀþþN High 112–582 þ þþþ þþþ C Low 329–626 ND þNþCND HD206AA þþþN High WT, wild type; ND, not determined. a Restriction was measured by infecting PMA-treated U937 cells stably expressing the indicated SAMHD1 variants with HIV-1-GFP.