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• W1991418438 abstract "SAMHD1, a dGTP-regulated deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase, down-regulates dNTP pools in terminally differentiated and quiescent cells, thereby inhibiting HIV-1 infection at the reverse transcription step. HIV-2 and simian immunodeficiency virus (SIV) counteract this restriction via a virion-associated virulence accessory factor, Vpx (Vpr in some SIVs), which loads SAMHD1 onto CRL4-DCAF1 E3 ubiquitin ligase for polyubiquitination, programming it for proteasome-dependent degradation. However, the detailed molecular mechanisms of SAMHD1 recruitment to the E3 ligase have not been defined. Further, whether divergent, orthologous Vpx proteins, encoded by distinct HIV/SIV strains, bind SAMHD1 in a similar manner, at a molecular level, is not known. We applied surface plasmon resonance analysis to assess the requirements for and kinetics of binding between various primate SAMHD1 proteins and Vpx proteins from SIV or HIV-2 strains. Our data indicate that Vpx proteins, bound to DCAF1, interface with the C terminus of primate SAMHD1 proteins with nanomolar affinity, manifested by rapid association and slow dissociation. Further, we provide evidence that Vpx binding to SAMHD1 inhibits its catalytic activity and induces disassembly of a dGTP-dependent oligomer. Our studies reveal a previously unrecognized biochemical mechanism of Vpx-mediated SAMHD1 inhibition: direct down-modulation of its catalytic activity, mediated by the same binding event that leads to SAMHD1 recruitment to the E3 ubiquitin ligase for proteasome-dependent degradation.Background: SAMHD1 is counteracted by the HIV-2/SIV virulence factor Vpx, which directs it for degradation.Results: Vpx in complex with DDB1-DCAF1 binds to the C terminus of SAMHD1, inhibits its catalytic activity, and dissociates SAMHD1 tetramers.Conclusion: The viral countermeasures by Vpx are manifested at multiple levels.Significance: Identifying molecular mechanisms of viral countermeasures against HIV restriction factors is important for designing antiviral therapeutics. SAMHD1, a dGTP-regulated deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase, down-regulates dNTP pools in terminally differentiated and quiescent cells, thereby inhibiting HIV-1 infection at the reverse transcription step. HIV-2 and simian immunodeficiency virus (SIV) counteract this restriction via a virion-associated virulence accessory factor, Vpx (Vpr in some SIVs), which loads SAMHD1 onto CRL4-DCAF1 E3 ubiquitin ligase for polyubiquitination, programming it for proteasome-dependent degradation. However, the detailed molecular mechanisms of SAMHD1 recruitment to the E3 ligase have not been defined. Further, whether divergent, orthologous Vpx proteins, encoded by distinct HIV/SIV strains, bind SAMHD1 in a similar manner, at a molecular level, is not known. We applied surface plasmon resonance analysis to assess the requirements for and kinetics of binding between various primate SAMHD1 proteins and Vpx proteins from SIV or HIV-2 strains. Our data indicate that Vpx proteins, bound to DCAF1, interface with the C terminus of primate SAMHD1 proteins with nanomolar affinity, manifested by rapid association and slow dissociation. Further, we provide evidence that Vpx binding to SAMHD1 inhibits its catalytic activity and induces disassembly of a dGTP-dependent oligomer. Our studies reveal a previously unrecognized biochemical mechanism of Vpx-mediated SAMHD1 inhibition: direct down-modulation of its catalytic activity, mediated by the same binding event that leads to SAMHD1 recruitment to the E3 ubiquitin ligase for proteasome-dependent degradation. Background: SAMHD1 is counteracted by the HIV-2/SIV virulence factor Vpx, which directs it for degradation. Results: Vpx in complex with DDB1-DCAF1 binds to the C terminus of SAMHD1, inhibits its catalytic activity, and dissociates SAMHD1 tetramers. Conclusion: The viral countermeasures by Vpx are manifested at multiple levels. Significance: Identifying molecular mechanisms of viral countermeasures against HIV restriction factors is important for designing antiviral therapeutics. SAMHD1 (sterile α motif and HD domain-containing protein 1) is an antiviral factor, inhibiting HIV/SIV 3The abbreviations used are:SIV, simian immunodeficiency virus; SAMHD1-FL, full-length SAMHD1; ΔN-SAMHD1, SAMHD1 residues 113–626; SAMHD1-ΔC, SAMHD1 residues 1–595; DCAF1CA, DCAF1 residues 1040–1400; DCAF1CB, DCAF1 residues 1045–1396; GST-Hu SAMHD1-FL, full-length human SAMHD1 expressed as a GST fusion protein; SAM, sterile α motif; RU, response units; SPR, surface plasmon resonance; CTD, C-terminal domain. infection of myeloid cells and quiescent CD4+ lymphocytes at the post-entry stage (1Berger A. Sommer A.F. Zwarg J. Hamdorf M. Welzel K. Esly N. Panitz S. Reuter A. Ramos I. Jatiani A. Mulder L.C. Fernandez-Sesma A. Rutsch F. Simon V. König R. Flory E. SAMHD1-deficient CD14+ cells from individuals with Aicardi-Goutieres syndrome are highly susceptible to HIV-1 infection.PLoS Pathog. 2011; 7: e1002425Crossref PubMed Scopus (206) Google Scholar, 2Hrecka K. Hao C. Gierszewska M. Swanson S.K. Kesik-Brodacka M. Srivastava S. Florens L. Washburn M.P. Skowronski J. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein.Nature. 2011; 474: 658-661Crossref PubMed Scopus (910) Google Scholar, 3Laguette N. Sobhian B. Casartelli N. Ringeard M. Chable-Bessia C. Ségéral E. Yatim A. Emiliani S. Schwartz O. Benkirane M. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx.Nature. 2011; 474: 654-657Crossref PubMed Scopus (1118) Google Scholar, 4Descours B. Cribier A. Chable-Bessia C. Ayinde D. Rice G. Crow Y. Yatim A. Schwartz O. Laguette N. Benkirane M. SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4+ T-cells.Retrovirology. 2012; 9: 87Crossref PubMed Scopus (263) Google Scholar, 5Baldauf H.M. Pan X. Erikson E. Schmidt S. Daddacha W. Burggraf M. Schenkova K. Ambiel I. Wabnitz G. Gramberg T. Panitz S. Flory E. Landau N.R. Sertel S. Rutsch F. Lasitschka F. Kim B. König R. Fackler O.T. Keppler O.T. SAMHD1 restricts HIV-1 infection in resting CD4+ T cells.Nat. Med. 2012; 18: 1682-1687Crossref PubMed Scopus (452) Google Scholar, 6St Gelais C. de Silva S. Amie S.M. Coleman C.M. Hoy H. Hollenbaugh J.A. Kim B. Wu L. SAMHD1 restricts HIV-1 infection in dendritic cells (DCs) by dNTP depletion, but its expression in DCs and primary CD4+ T-lymphocytes cannot be upregulated by interferons.Retrovirology. 2012; 9: 105Crossref PubMed Scopus (151) Google Scholar). It also prevents direct transmission of HIV-1 from infected T lymphocytes to monocyte-derived dendritic cells (7Puigdomènech I. Casartelli N. Porrot F. Schwartz O. SAMHD1 restricts HIV-1 cell-to-cell transmission and limits immune detection in monocyte-derived dendritic cells.J. Virol. 2013; 87: 2846-2856Crossref PubMed Scopus (53) Google Scholar). SAMHD1 is a deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase, the catalytic activity of which is regulated by dGTP binding at an allosteric site (8Goldstone D.C. Ennis-Adeniran V. Hedden J.J. Groom H.C. Rice G.I. Christodoulou E. Walker P.A. Kelly G. Haire L.F. Yap M.W. de Carvalho L.P. Stoye J.P. Crow Y.J. Taylor I.A. Webb M. HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase.Nature. 2011; 480: 379-382Crossref PubMed Scopus (609) Google Scholar, 9Powell R.D. Holland P.J. Hollis T. Perrino F.W. Aicardi-Goutieres syndrome gene and HIV-1 restriction factor SAMHD1 is a dGTP-regulated deoxynucleotide triphosphohydrolase.J. Biol. Chem. 2011; 286: 43596-43600Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Current models suggest that SAMHD1 maintains the cellular dNTP pools at low levels in those cells, thereby blocking viral reverse transcriptase activity (10Kim B. Nguyen L.A. Daddacha W. Hollenbaugh J.A. Tight interplay among SAMHD1 protein level, cellular dNTP levels, and HIV-1 proviral DNA synthesis kinetics in human primary monocyte-derived macrophages.J. Biol. Chem. 2012; 287: 21570-21574Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 11Lahouassa H. Daddacha W. Hofmann H. Ayinde D. Logue E.C. Dragin L. Bloch N. Maudet C. Bertrand M. Gramberg T. Pancino G. Priet S. Canard B. Laguette N. Benkirane M. Transy C. Landau N.R. Kim B. Margottin-Goguet F. SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates.Nat. Immunol. 2012; 13: 223-228Crossref PubMed Scopus (617) Google Scholar). SAMHD1 appears to have broad antiviral activity against diverse retroviruses (12White T.E. Brañdariz-Nunez A. Valle-Casuso J.C. Amie S. Nguyen L. Kim B. Brojatsch J. Diaz-Griffero F. Contribution of SAM and HD domains to retroviral restriction mediated by human SAMHD1.Virology. 2013; 436: 81-90Crossref PubMed Scopus (103) Google Scholar, 13Gramberg T. Kahle T. Bloch N. Wittmann S. Müllers E. Daddacha W. Hofmann H. Kim B. Lindemann D. Landau N.R. Restriction of diverse retroviruses by SAMHD1.Retrovirology. 2013; 10: 26Crossref PubMed Scopus (113) Google Scholar). SAMHD1 comprises two structural domains: a sterile α motif (SAM) domain and a dNTPase domain, which encompasses a metal-dependent phosphohydrolase homologous region with a conserved histidine and aspartate (HD) motif. These two domains are connected by a short linker and flanked by unstructured regions (Fig. 1A). The N terminus, preceding the SAM domain, contains a nuclear localization signal (14Brandariz-Nuñez A. Valle-Casuso J.C. White T.E. Laguette N. Benkirane M. Brojatsch J. Diaz-Griffero F. Role of SAMHD1 nuclear localization in restriction of HIV-1 and SIVmac.Retrovirology. 2012; 9: 49Crossref PubMed Scopus (116) Google Scholar, 15Hofmann H. Logue E.C. Bloch N. Daddacha W. Polsky S.B. Schultz M.L. Kim B. Landau N.R. The Vpx lentiviral accessory protein targets SAMHD1 for degradation in the nucleus.J. Virol. 2012; 86: 12552-12560Crossref PubMed Scopus (102) Google Scholar, 16Wei W. Guo H. Han X. Liu X. Zhou X. Zhang W. Yu X.F. A novel DCAF1-binding motif required for Vpx-mediated degradation of nuclear SAMHD1 and Vpr-induced G2 arrest.Cell. Microbiol. 2012; 14: 1745-1756Crossref PubMed Scopus (44) Google Scholar). The crystal structure of the dNTPase domain has been determined and suggests, along with biochemical studies, that dGTP binding at an allosteric site, formed by two monomers, regulates its dNTPase activity (8Goldstone D.C. Ennis-Adeniran V. Hedden J.J. Groom H.C. Rice G.I. Christodoulou E. Walker P.A. Kelly G. Haire L.F. Yap M.W. de Carvalho L.P. Stoye J.P. Crow Y.J. Taylor I.A. Webb M. HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase.Nature. 2011; 480: 379-382Crossref PubMed Scopus (609) Google Scholar). The same domain also contains exonuclease activity with RNA binding affinity (12White T.E. Brañdariz-Nunez A. Valle-Casuso J.C. Amie S. Nguyen L. Kim B. Brojatsch J. Diaz-Griffero F. Contribution of SAM and HD domains to retroviral restriction mediated by human SAMHD1.Virology. 2013; 436: 81-90Crossref PubMed Scopus (103) Google Scholar, 17Beloglazova N. Flick R. Tchigvintsev A. Brown G. Popovic A. Nocek B. Yakunin A.F. Nuclease activity of the human SAMHD1 protein implicated in the Aicardi-Goutieres syndrome and HIV-1 restriction.J. Biol. Chem. 2013; 288: 8101-8110Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 18Goncalves A. Karayel E. Rice G.I. Bennett K.L. Crow Y.J. Superti-Furga G. Bürckstümmer T. SAMHD1 is a nucleic-acid binding protein that is mislocalized due to aicardi-goutieres syndrome-associated mutations.Hum. Mutat. 2012; 33: 1116-1122Crossref PubMed Scopus (107) Google Scholar). We recently provided biochemical and virological evidence that the biologically active form of human SAMHD1 is a tetramer and that its C terminus is required for efficient depletion of dNTP pools and inhibition of HIV-1 infection in monocytes (19Yan J. Kaur S Delucia M Hao C Mehrens J Wang C Golczak M Palczewski K Gronenborn AM Ahn J Skowronski J Tetramerization of SAMHD1 is required for biological activity and inhibition of HIV infection.J. Biol. Chem. 2013; 288: 10406-10417Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Similar to other restriction factors, such as APOBEC3G and tetherin, SAMHD1 restriction is counteracted by an HIV/SIV accessory virulence factor, specifically Vpx, which is encoded by HIV-2 and SIV. Vpx binds DCAF1 (DDB1- and CUL4-associated factor 1), a substrate receptor for the CRL4 (Cullin4 RING ubiquitin ligase) E3 ubiquitin ligase, and recruits SAMHD1 to the E3 ligase for proteasome-dependent degradation (1Berger A. Sommer A.F. Zwarg J. Hamdorf M. Welzel K. Esly N. Panitz S. Reuter A. Ramos I. Jatiani A. Mulder L.C. Fernandez-Sesma A. Rutsch F. Simon V. König R. Flory E. SAMHD1-deficient CD14+ cells from individuals with Aicardi-Goutieres syndrome are highly susceptible to HIV-1 infection.PLoS Pathog. 2011; 7: e1002425Crossref PubMed Scopus (206) Google Scholar, 2Hrecka K. Hao C. Gierszewska M. Swanson S.K. Kesik-Brodacka M. Srivastava S. Florens L. Washburn M.P. Skowronski J. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein.Nature. 2011; 474: 658-661Crossref PubMed Scopus (910) Google Scholar, 3Laguette N. Sobhian B. Casartelli N. Ringeard M. Chable-Bessia C. Ségéral E. Yatim A. Emiliani S. Schwartz O. Benkirane M. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx.Nature. 2011; 474: 654-657Crossref PubMed Scopus (1118) Google Scholar, 20Ahn J. Hao C. Yan J. DeLucia M. Mehrens J. Wang C. Gronenborn A.M. Skowronski J. HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.J. Biol. Chem. 2012; 287: 12550-12558Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The discovery of an interplay between the virulence factor and these host cell factors resolved a longstanding unanswered observation; Vpx facilitates transduction of dendritic cells and macrophages and relieves the inhibition of HIV-1 infection in restricting cells (21Yu X.F. Yu Q.C. Essex M. Lee T.H. The vpx gene of simian immunodeficiency virus facilitates efficient viral replication in fresh lymphocytes and macrophage.J. Virol. 1991; 65: 5088-5091Crossref PubMed Google Scholar, 22Kawamura M. Sakai H. Adachi A. Human immunodeficiency virus Vpx is required for the early phase of replication in peripheral blood mononuclear cells.Microbiol. Immunol. 1994; 38: 871-878Crossref PubMed Scopus (46) Google Scholar, 23Hirsch V.M. Sharkey M.E. Brown C.R. Brichacek B. Goldstein S. Wakefield J. Byrum R. Elkins W.R. Hahn B.H. Lifson J.D. Stevenson M. Vpx is required for dissemination and pathogenesis of SIV(SM) PBj. Evidence of macrophage-dependent viral amplification.Nat. Med. 1998; 4: 1401-1408Crossref PubMed Scopus (154) Google Scholar, 24Goujon C. Rivière L. Jarrosson-Wuilleme L. Bernaud J. Rigal D. Darlix J.L. Cimarelli A. SIVSM/HIV-2 Vpx proteins promote retroviral escape from a proteasome-dependent restriction pathway present in human dendritic cells.Retrovirology. 2007; 4: 2Crossref PubMed Scopus (170) Google Scholar). Phylogenetic tree and functional analyses of Vpx and its homolog, Vpr, combined with similar analyses of SAMHD1, indicate that these virulence accessory factors and the host restriction factor have undergone an evolutionary arms race (25Zhang C. de Silva S. Wang J.H. Wu L. Co-evolution of primate SAMHD1 and lentivirus Vpx leads to the loss of the vpx gene in HIV-1 ancestor.PloS One. 2012; 7: e37477Crossref PubMed Scopus (37) Google Scholar, 26Lim E.S. Fregoso O.I. McCoy C.O. Matsen F.A. Malik H.S. Emerman M. The ability of primate lentiviruses to degrade the monocyte restriction factor SAMHD1 preceded the birth of the viral accessory protein Vpx.Cell Host Microbe. 2012; 11: 194-204Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 27Laguette N. Rahm N. Sobhian B. Chable-Bessia C. Münch J. Snoeck J. Sauter D. Switzer W.M. Heneine W. Kirchhoff F. Delsuc F. Telenti A. Benkirane M. Evolutionary and functional analyses of the interaction between the myeloid restriction factor SAMHD1 and the lentiviral Vpx protein.Cell Host Microbe. 2012; 11: 205-217Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Interestingly, two distinct regions of SAMHD1, the SAM domain, at the N terminus, and the C terminus, distal to the dNTPase domain, display strong positive selection during primate evolution. However, the detailed molecular mechanisms by which Vpx recruits SAMHD1 to CRL4-DCAF1 are yet to be specified. Here, we show that HIV-2 and SIVmac (isolated from Macaque monkey) Vpx recruit human and several simian SAMHD1 proteins to the CRL4-DCAF1 E3 ubiquitin ligase by interacting with the highly conserved C terminus of SAMHD1. Further, we show that a highly conserved, specific sequence motif at the Vpx N terminus is essential for efficient recruitment of SAMHD1. Real-time binding assays, using surface plasmon resonance (SPR) analysis, suggest that Vpx significantly increases the association rate and decreases the dissociation rate of SAMHD1 binding to the substrate adaptor-receptor complex of the E3 ubiquitin ligase. Surprisingly, Vpx-mediated recruitment of SAMHD1 to the substrate adaptor-receptor complex deactivates its dNTPase catalysis and subsequently disassembles dGTP-dependent tetramers to dimer and monomers. These results suggest that Vpx employs an additional viral countermeasure, prior to and independently of proteasome-dependent down-regulation of SAMHD1. The cDNAs encoding full-length SAMHD1 (SAMHD1-FL) or residues 113–626 (ΔN-SAMHD1) or residues 1–595 (SAMHD1-ΔC) of human, rhesus, or De Brazza's SAMHD1 were cloned into pCDNA3.1 (Invitrogen) with an HA tag at its N terminus or into pET28 (EMD Biosciences) with His6 and T7 tags at its N terminus. The cDNAs encoding SAMHD1 from other primates, including African green monkey, mandrill, red capped monkey, and sooty mangabey were also cloned into the pET28 vector. SAMHD1 full-length or the C terminus (residue 595–626) was also cloned into pET41 (EMD Biosciences) with N-terminal GST and His6 tags. N-terminally HA-tagged full-length DCAF1 or DCAF1 C-terminal residues 1040–1400 (DCAF1CA) were also cloned into pCDNA3.1 vector. The SIVmac, HIV-2 Rod9, and HIV-2 7312a Vpx (VpxSIVmac, VpxRod9, and Vpx7312a, respectively) were cloned into pCDNA3.1 with either an HA or V5 tag at its N terminus or into pET43 vectors with a C-terminal His6 tag, modified to include a tobacco etch virus protease site at the C terminus of a NusA fusion protein. The cDNAs encoding VpxSIVmac and VpxRod9 residues 1–102 or Vpx7312a residues 1–101 (Vpx(ΔC)SIVmac, Vpx(ΔC)Rod9, and Vpx(ΔC)7312a, respectively) were also cloned into the modified pET43 vectors. The cDNAs for monkey SAMHD1 and VpxRod9 and Vpx7312a were provided by M. Emerman (Fred Hutchison Cancer Center, Seattle, WA). Site-specific mutants of Vpx and SAMHD1 were prepared using QuikChange mutagenesis kits (Agilent). The various SAMHD1 and NusA-Vpx fusion proteins were expressed in Escherichia coli Rosetta 2 (DE3) cultured in Luria-Bertani medium with 0.4 mm isopropyl 1-thio-β-d-galactopyranoside at 18 °C for 16 h. Proteins were first purified using a 5-ml nickel-nitrilotriacetic acid column (GE Healthcare), and then the aggregates were removed by gel filtration column chromatography (Hi-Load Superdex200 16/60, GE Healthcare) equilibrated with a buffer containing 25 mm sodium phosphate, pH 7.5, 150 mm NaCl, 2 mm DTT, 10% glycerol, and 0.02% sodium azide. The DDB1-DCAF1CB (DCAF1 residues 1045–1396) complex was expressed and purified from SF21 cells co-infected with recombinant bacuolviruses at a multiplicity of infection of 2 for 40 h, as described previously (20Ahn J. Hao C. Yan J. DeLucia M. Mehrens J. Wang C. Gronenborn A.M. Skowronski J. HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.J. Biol. Chem. 2012; 287: 12550-12558Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). For preparation of multiprotein complexes, DDB1-DCAF1CB and NusA-Vpx proteins were mixed at a molar ratio of 1:3, digested with tobacco etch virus protease, and purified over an 8-ml MONO Q column (GE Healthcare) at pH 7.5, using a 0–1 m NaCl gradient. All other proteins were prepared as described previously (20Ahn J. Hao C. Yan J. DeLucia M. Mehrens J. Wang C. Gronenborn A.M. Skowronski J. HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.J. Biol. Chem. 2012; 287: 12550-12558Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 28Ahn J. Vu T. Novince Z. Guerrero-Santoro J. Rapic-Otrin V. Gronenborn A.M. HIV-1 Vpr loads uracil DNA glycosylase-2 onto DCAF1, a substrate recognition subunit of a cullin 4A-ring E3 ubiquitin ligase for proteasome-dependent degradation.J. Biol. Chem. 2010; 285: 37333-37341Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Human embryonic kidney cell lines (HEK293 from ATCC) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum with antibiotics. Cells were plated on 6-cm plates, 24 h prior to transient transfection, and grown to 90–95% confluence. HEK293 cells were transfected with 7 μg of a mixture of pCNDA3.1 plasmids encoding specific cDNAs, as indicated, using LipofectAMINE 2000 (Invitrogen), according to the manufacturer's protocol. When indicated, the transfected cells were treated with 25 μm MG132 (Boston Biochem) for 6 h, 42 h after transfection. Transfected cells were harvested and treated with 300 μl of lysis buffer containing 25 mm sodium phosphate, pH 7.5, 300 mm NaCl, 1 mm EDTA, 0.3% Nonidet P-40, 1% Tween, 5% glycerol, and 1 mm phenylmethylsulfonyl fluoride. Proteins in the lysate were separated by 4–20% gradient SDS-PAGE, transferred to PVDF membrane, and subsequently identified by immunoblotting. For detection of proteins, anti-HA (Covance), anti-V5 (Sigma), and anti-actin (Sigma) antibodies were used. SAMHD1 proteins (0.5 μm) or their mixtures with dGTP (200 μm) in 25 mm sodium phosphate, pH 7.5, 150 mm NaCl, 5% glycerol, 1 mm DTT, and 0.02% sodium azide were injected into a 24-ml analytical Superdex200 column (10 × 300 mm, GE Healthcare) at a flow rate of 0.8 ml/min, equilibrated with the same buffer lacking the reducing agent. The elution profiles were recorded by monitoring fluorescence, with excitation at 282 nm and emission at 313 nm. SPR experiments were performed using a BIAcore 2000 instrument (GE Healthcare) at 12 °C. CM5 sensor chips were coated with anti-GST antibodies according to the manufacturer's protocol (GST capture kit, GE Healthcare). GST-SAMHD1 variants, at a concentration of 10 nm in running buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.005% P20, 0.02% sodium azide, 1 mm tris(2-carboxyethyl)phosphine), were injected into the flow cell at a rate of 5 μl/min until the increase in response units (RU) reached 50–60 RU. The first flow cell was used as an in-line reference with GST as ligand. The analytes, at different concentrations in the running buffer, were injected into the flow cells for 2 min at a flow rate of 30 μl/min, followed by a 3-min dissociation phase. The sensor surface was regenerated by repeated injections of a regeneration buffer containing 10 mm sodium acetate, pH 5.0. For steady-state analysis, the equilibrium response of each injection at 120 s was plotted against the concentration of injected protein, using a non-linear, one-site nonspecific model (GraphPad Prism 5, GraphPad Software) to obtain the equilibrium dissociation constant (Kd) and binding response maximum (Rmax). Non-equilibrium data, including the rate of association, kon, were globally fit to a predefined one-state model using BIAevaluation software (version 4.1). Typically, E1 (UBA1, 0.2 μm), E2 (UbcH5b, 2.5 μm), and E3 complexes (mixtures of equimolar amounts of DDB1-DCAF1CB-VpxSIVmac, -Vpx7312a, or -VpxRod9 and CUL4A-RBX1 at 0.3 μm) were incubated with 0.6 μm SAMHD1 (SAMHD1-FL or SAMHD1-ΔC) and 2.5 μm ubiquitin, in a buffer containing 10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5% glycerol, 20 units/ml pyrophosphatase, 2 mm DTT, and 5 mm ATP at 37 °C for the indicated times. The extent of ubiquitination was assessed by immunoblotting with anti-T7 antibody (EMD Biosciences) after separation of reaction mixtures on 4–20% gradient SDS-PAGE and transfer to PVDF membrane. Typically, 60 μl of SAMHD1 proteins (1.6 μm) were mixed with an equal volume of dGTP (100 μm) in a buffer containing 25 mm sodium phosphate, pH 7.5, 50 mm NaCl, 5% glycerol, 2 mm MgCl2, and 0.02% sodium azide. After 60 s, the mixtures were diluted with 3.13 mm glutaraldehyde, in the same buffer, and incubated for 3 min before cross-linking reactions (50 μl) were quenched with 290 mm Tris-HCl, pH 6.8 (20 μl). The resulting reactions were separated by 4–20% SDS-PAGE and visualized with Coomassie Brilliant Blue staining. Assays of SAMHD1 dGTP-dependent enzymatic activities were carried out in a reaction buffer containing 20 mm Tris-HCl, pH 7.8, 50 mm NaCl, 2 mm MgCl2, 5% glycerol, an appropriate concentration of dNTP (0–100 μm), and 0.1 μm recombinant SAMHD1. The reactions were stopped by mixing a 50-μl enzymatic reaction with 20 μl of 70 mm EDTA, after a specific time interval. Quenched reactions (50 μl) were injected into a Capcell Pak C18 reversed-phase column (4.6 × 250 mm; Phenomenex), pre-equilibrated with a buffer containing 10 mm ammonium phosphate, pH 7.8, and 4.8% methanol. Deoxyribonucleosides and dNTPs were eluted with a linear gradient (4.8–19.2%) of methanol over 22.5 min at a flow rate of 1.5 ml/min. The amounts of products were quantified by peak integration of the absorbance trace at UV 260 nm and converted to moles based on the calibration curve of deoxyribonucleosides. 100 μl of protein solution, typically at ∼2 mg/ml, was injected into an analytical Superdex200 column (10 × 300 mm; GE Healthcare) with in-line multiangle light scattering (HELEOS, Wyatt Technology), variable wavelength UV detector (Agilent 1100, Agilent Technologies), and refractive index detector (Optilab rEX, Wyatt Technology) at a flow rate of 0.5 ml/min in a buffer containing 25 mm sodium phosphate, pH 7.5, 50–150 mm NaCl, and 0.02% sodium azide. The molecular masses of eluted protein species were determined using the ASTRA version 5.3.4 program (Wyatt Technologies). We previously described that SIVmac Vpx facilitates loading of human SAMHD1 onto the substrate adaptor-receptor complex (DDB1-DCAF1) of the CRL4 E3 ubiquitin ligase for proteasome-dependent degradation (20Ahn J. Hao C. Yan J. DeLucia M. Mehrens J. Wang C. Gronenborn A.M. Skowronski J. HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.J. Biol. Chem. 2012; 287: 12550-12558Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). We also identified critical residues at the recruitment interfaces. Specifically, C-terminal SAMHD1 residues Arg-617, Leu-620, and Phe-621, distal to the dNTPase domain, are essential for binding (Fig. 1A). The alignment of other monkey SAMHD1 sequences shows strict conservation of this C-terminal region (supplemental Fig. 1) (27Laguette N. Rahm N. Sobhian B. Chable-Bessia C. Münch J. Snoeck J. Sauter D. Switzer W.M. Heneine W. Kirchhoff F. Delsuc F. Telenti A. Benkirane M. Evolutionary and functional analyses of the interaction between the myeloid restriction factor SAMHD1 and the lentiviral Vpx protein.Cell Host Microbe. 2012; 11: 205-217Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Sequence alignment of Vpx proteins from SIVmac and HIV-2 indicates conservation of four N-terminal residues (Asn-12, Glu-15, Glu-16, and Thr-17 for VpxSIVmac and VpxRod9 and Asn-11, Glu-14, Glu-15, and Thr-16 for Vpx7312a) that were previously shown to be at the SAMDH1 recruitment interface (Fig. 1B) (20Ahn J. Hao C. Yan J. DeLucia M. Mehrens J. Wang C. Gronenborn A.M. Skowronski J. HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.J. Biol. Chem. 2012; 287: 12550-12558Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Based on these observations, SIVmac and HIV-2 Vpx proteins probably bind the C-terminal sequences of primate SAMHD1 proteins in a similar manner. To gain mechanistic insight into how Vpx facilitates SAMHD1 binding to DDB1-DCAF1, we developed an SPR-based assay to evaluate the rates of association and dissociation between SAMHD1 and DDB1-DCAF1CB (residues 1045–1396) in complex with Vpx. Specifically, full-length human SAMHD1 was expressed as a GST fusion protein (GST-Hu SAMHD1-FL) and immobilized onto a CM5 chip coated with anti-GST antibody. Then increasing concentrations of DDB1-DCAF1CB-Vpx(ΔC)SIVmac were applied, and sensorgrams were recorded. Surprisingly, DDB1-DCAF1CB-Vpx(ΔC)SIVmac dissociation from human SAMHD1 was negligible for 180 s after the injection was complete (Fig. 2A). Similar observations were made with immobilized rhesus (Fig. 2B) and De Brazza's SAMHD1 (data not shown). In fact, the rate of association (kon) and the dissociation constant (Kd) were essentially the same for all three SAMHD1 proteins, ranging from 18 to 34 × 103 m−1 s−1 and from 213 to 521 nm, respectively (Table 1 and Fig. 2C). Similar SPR experiments showed that SAMHD1 did not bind to the DDB1-DCAF1CB complex in the a" @default.
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• W1991418438 title "HIV-2 and SIVmac Accessory Virulence Factor Vpx Down-regulates SAMHD1 Enzyme Catalysis Prior to Proteasome-dependent Degradation" @default.
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