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• W2216889613 abstract "Mechanisms underlying HIV-1 latency remain among the most crucial questions that need to be answered to adopt strategies for purging the latent viral reservoirs. Here we show that HIV-1 accessory protein Vpr induces depletion of class I HDACs, including HDAC1, 2, 3, and 8, to overcome latency in macrophages. We found that Vpr binds and depletes chromatin-associated class I HDACs through a VprBP-dependent mechanism, with HDAC3 as the most affected class I HDAC. De novo expression of Vpr in infected macrophages induced depletion of HDAC1 and 3 on the HIV-1 LTR that was associated with hyperacetylation of histones on the HIV-1 LTR. As a result of hyperacetylation of histones on HIV-1 promotor, the virus established an active promotor and this contributed to the acute infection of macrophages. Collectively, HIV-1 Vpr down-regulates class I HDACs on chromatin to counteract latent infections of macrophages. Mechanisms underlying HIV-1 latency remain among the most crucial questions that need to be answered to adopt strategies for purging the latent viral reservoirs. Here we show that HIV-1 accessory protein Vpr induces depletion of class I HDACs, including HDAC1, 2, 3, and 8, to overcome latency in macrophages. We found that Vpr binds and depletes chromatin-associated class I HDACs through a VprBP-dependent mechanism, with HDAC3 as the most affected class I HDAC. De novo expression of Vpr in infected macrophages induced depletion of HDAC1 and 3 on the HIV-1 LTR that was associated with hyperacetylation of histones on the HIV-1 LTR. As a result of hyperacetylation of histones on HIV-1 promotor, the virus established an active promotor and this contributed to the acute infection of macrophages. Collectively, HIV-1 Vpr down-regulates class I HDACs on chromatin to counteract latent infections of macrophages. HIV-1 genome encodes 4 accessory proteins, including Nef, Vif, Vpu, and Vpr. These proteins are known to down-regulate cellular proteins through different mechanisms to facilitate various stages of viral infection. For instance, Nef down-regulates surface expression of CD4 and MHC-I (1Mangasarian A. Piguet V. Wang J.K. Chen Y.L. Trono D. Nef-induced CD4 and major histocompatibility complex class I (MHC-I) downregulation are governed by distinct determinants: N-terminal α-helix and proline repeat of nef selectively regulate MHC-I trafficking.J. Virol. 1999; 73: 1964-1973Crossref PubMed Google Scholar); Vif down-regulates APOBEC3G and APOBEC3F (2Liu B. Sarkis P.T. Luo K. Yu Y. Yu X.F. Regulation of APOBEC3F and human immunodeficiency virus type 1 Vif by Vif-Cul5-ElonB/C ubiquitin ligase.J. Virol. 2005; 79: 9579-9587Crossref PubMed Scopus (134) Google Scholar), and Vpu down-regulates surface CD4 and tetherin (3Pickering S. Hué S. Kim E.Y. Reddy S. Wolinsky S.M. Neil S.j.D. Preservation of thetherin and CD4 counteractivities in circulating Vpu alleles despite extensive sequence variation whithin HIV-1 infected individuals.Plos. Pathog. 2014; 10: e1003895Crossref PubMed Scopus (46) Google Scholar). Recent studies also show a growing list of cellular proteins whose degradation is induced by Vpr while the outcomes of their degradation remain to be elucidated. HIV-1 Vpr is well-documented to interact with the Cul4-DDB1[VprBP] E3 ubiquitin ligase to direct proteasomal degradation of a number of cellular proteins and induce G2/M cell cycle arrest (4Ahn 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 Scholar5Hrecka K. Gierszewska M. Srivastava S. Kozaczkiewicz L. Swanson S.K. Florens L. Washburn M.P. Skowronski J. Lentiviral Vpr usurps Cul4–DDB1[VprBP] E3 ubiquitin ligase to modulate cell cycle.Pro. Nat. Aca. Sci. U.S.A. 2007; 104: 11778-11783Crossref PubMed Scopus (188) Google Scholar, 6Romani B. Engelbrecht S. Human immunodeficiency virus type 1 Vpr: functions and molecular interactions.J. Gen. Virol. 2009; 90: 1795-1805Crossref PubMed Scopus (39) Google Scholar7Tan L. Ehrlich E. Yu X.-F. DDB1 and Cul4A are required for human immunodeficiency virus type 1 Vpr-induced G2 arrest.J. Virol. 2007; 81: 10822-10830Crossref PubMed Scopus (106) Google Scholar). Vpr enhances proteasomal degradation of the natural substrates of the Cul4-DDB1[VprBP] E3 ubiquitin ligase (8Hrecka 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 (912) Google Scholar, 9Laguette 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 (1121) Google Scholar10Romani B. Shaykh-Baygloo N. Aghasadeghi M.R. Allahbakhshi E. HIV-1 Vpr protein enhances proteasomal degradation of MCM10 DNA replication factor through the Cul4-DDB1[VprBP] E3 ubiquitin ligase to induce G2/M cell cycle arrest.J. Biol. Chem. 2015; 290: 17380-17389Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). For instance, Vpr has been shown to enhance proteasomal degradation of UNG2 and dicer (11Wen X. Casey Klockow L. Nekorchuk M. Sharifi H.J. de-Noronha C.M.C. The HIV1 protein Vpr acts to enhance constitutive DCAF1-dependent UNG2 turnover.PLoS ONE. 2012; 7: e30939Crossref PubMed Scopus (29) Google Scholar, 12Casey Klockow L. Sharifi H.J. Wen X. Flagg M. Furuya A.K.M. Nekorchuk M. de-Noronha C.M.C. The HIV-1 protein Vpr targets the endoribonuclease Dicer for proteasomal degradation to boost macrophage infection.Virology. 2013; 444: 191-202Crossref PubMed Scopus (40) Google Scholar). These proteins are recruited by Vpr-binding protein (VprBP) 3The abbreviations used are: VprBP, Vpr-binding protein; HDAC, histone deacetylase; MDM, monocyte-derived macrophage. to the E3 ubiquitin ligase complexes as their natural substrates. By binding to VprBP, Vpr enhances degradation of the natural substrates recruited by VprBP (10Romani B. Shaykh-Baygloo N. Aghasadeghi M.R. Allahbakhshi E. HIV-1 Vpr protein enhances proteasomal degradation of MCM10 DNA replication factor through the Cul4-DDB1[VprBP] E3 ubiquitin ligase to induce G2/M cell cycle arrest.J. Biol. Chem. 2015; 290: 17380-17389Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 13Wang X. Singh S. Jung H.Y. Yang G. Jun S. Sastry K.J. Park J.I. HIV-1 Vpr protein inhibits telomerase activity via the EDD-DDB1-VPRBP E3 ligase complex.J. Biol. Chem. 2013; 288: 15474-15480Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Furthermore, minichromosome maintenance complex component 10 (MCM10) was recently shown to be the natural substrate of the Cul4-DDB1[VprBP] E3 ubiquitin ligase (14Kaur M. Khan M. Kar A. Sharma A. Saxena S. CRL4-DDB1-VPRBP ubiquitin ligase mediates the stress triggered proteolysis of Mcm10.Nucleic Acids Res. 2012; 40: 7332-7346Crossref PubMed Scopus (36) Google Scholar) and our laboratory showed that proteasomal degradation of MCM10, is also enhanced by Vpr (10Romani B. Shaykh-Baygloo N. Aghasadeghi M.R. Allahbakhshi E. HIV-1 Vpr protein enhances proteasomal degradation of MCM10 DNA replication factor through the Cul4-DDB1[VprBP] E3 ubiquitin ligase to induce G2/M cell cycle arrest.J. Biol. Chem. 2015; 290: 17380-17389Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). It is believed that Vpr induces most of its functions by interacting with VprBP. To investigate the biological roles of Vpr, we investigated proteins that interact with VprBP. It was recently shown that VprBP directly interacts with nucleosomes by recognizing the unacetylated N-terminal tail of histone H3 (15Kim K. Heo K. Choi J. Jackson S. Kim H. Xiong Y. An W. Vpr-binding protein antagonizes p53-mediated transcription via direct interaction with H3 tail.Mol. Cell Biol. 2012; 32: 783-796Crossref PubMed Scopus (29) Google Scholar). In this complex, VprBP was also shown to bind histone deacetylase 1 (HDAC1). HDACs are a class of enzymes that remove acetyl groups from ϵ-N-acetyl lysine amino-acids of histones and other proteins and also regulate a number of key cellular processes, including gene expression, protein complex formation, and protein stability and localization (16Gallinari P. Di-Marco S. Jones P. Pallaoro M. Steinkühler C. HDACs, histone deacetylation and gene transcription: from molecular biology to cancer therapeutics.Cell Res. 2007; 17: 195-211Crossref PubMed Scopus (423) Google Scholar17Choudhary C. Kumar C. Gnad F. Nielsen M.L. Rehman M. Walther T.C. Olsen J.V. Mann M. Lysine acetylation targets protein complexes and co-regulates major cellular functions.Science. 2009; 325: 834-840Crossref PubMed Scopus (3150) Google Scholar, 18Ropero S. Esteller M. The role of histone deacetylases (HDACs) in human cancer.Mol. Oncol. 2007; 1: 19-25Crossref PubMed Scopus (726) Google Scholar19Yang X.J. Seto E. HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention.Oncogene. 2007; 26: 5310-5318Crossref PubMed Scopus (767) Google Scholar). In mammals, 18 HDACs have been described and they are classified into 4 classes based on their phylogeny and function (20Wang Z. Zang C. Cui K. Schones D.E. Barski A. Peng W. Zhao K. Genome-wide mapping of HATs and HDACs reveals distinct functions in active and inactive genes.Cell. 2009; 138: 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 21Xu W.S. Parmigiani R.B. Marks P.A. Histone deacetylase inhibitors: molecular mechanisms of action.Oncogene. 2007; 26: 5541-5552Crossref PubMed Scopus (1249) Google Scholar). There is a high level of homology among the members of class I HDACs, which comprises HDAC1, 2, 3, and 8. Class I HDACs localize to the nucleus, except for HDAC3, which can be found both in the cytoplasm and the nucleus (22Delcuve G.P. Khan D.H. Davie J.R. Roles of histone deacetylases in epigenetic regulation: emerging paradigms from studies with inhibitors.Clinical Epigenetics. 2012; 4: 5Crossref PubMed Scopus (350) Google Scholar, 23Lezin A. Gillet N. Olindo S. Signate A. Grandvaux N. Verlaeten O. Belrose G. Bittencourt M. d.-C. Hiscott J. Asquith B. Burny A. Smadja D. Cesaire R. Willems L. Histone deacetylase-mediated transcriptional activation reduces proviral loads in HTLV-1-associated myelopathy/tropical spastic paraparesis patients.Blood. 2007; 110: 10Crossref Scopus (74) Google Scholar). In this study, we found that Vpr binds and depletes chromatin-associated class I HDACs, enabling the virus to overcome latent infection in primary macrophages. HEK293T and HeLa cells were obtained from ATCC. TZM-bl cells were obtained from the NIH AIDS Research and Reference Reagent Program. Doxycycline-inducible HeLa cell lines (HeLa-iFlag-Vpr, HeLa-iFlag-Q65R) and the control cell line (HeLa-iMock) were described previously (10Romani B. Shaykh-Baygloo N. Aghasadeghi M.R. Allahbakhshi E. HIV-1 Vpr protein enhances proteasomal degradation of MCM10 DNA replication factor through the Cul4-DDB1[VprBP] E3 ubiquitin ligase to induce G2/M cell cycle arrest.J. Biol. Chem. 2015; 290: 17380-17389Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Expression of Flag-Vpr was induced by adding 1 μg/ml doxycycline. HeLa and the inducible HeLa cell lines were maintained in DMEM supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml of streptomycin. To isolate monocyte-derived macrophages (MDMs), hepatitis B virus and HIV-1-seronegative adult donors that had signed a consent form, provided peripheral blood samples. PBMCs were isolated from whole blood using Ficoll-density gradient centrifugation. Monocytes were primarily isolated by plastic adherence. For differentiation into mature macrophages, monocytes were cultured in RPMI supplemented with 50 ng/ml macrophage-colony stimulating factor M-CSF, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% FBS. After 7 days, cells were washed with PBS to rinse off the nonadherent cells. Purity of MDMs was verified by flow cytometry using antibodies against CD14 and CD16. HDAC1 (ab7028), HDAC2 (ab7029), HDAC3 (ab32369), HDAC8 (ab18968), histone H3 (ab70550), DDB1 (ab124672), p24 (ab9071), SIRT3 (ab86671), and HDAC4 (ab11968) antibodies were purchased from Abcam. GFP antibody (G6539), Flag antibody (F3165), Monoclonal Anti-HA-agarose beads, and ANTI-FLAG M2 affinity gel were from Sigma-Aldrich. GAPDH (14C10) and rabbit IgG isotype control (2729) were from Cell Signaling Technology. VprBP (A301–888A) antibody was from Bethyl Laboratories. Vpr (NP_057852) antibody was from Proteintech. Ace H3K9 (39917) and Ace H4K5 (39699) antibodies were from active motif. PE-Texas Red anti-human CD14 (MHCD1417) was from Life Technologies. APC/Cy7 anti-human CD16 antibody (302018) was from BioLegend. Mouse and rabbit HRP-conjugated antibodies were from Abcam. Protein A Sepharose beads were from Amersham Biosciences. Benzonase nuclease was from Novagen. Flag peptides, caffeine, ethidium bromide, doxycycline, SAHA, and DMSO were from Sigma-Aldrich. MG132 was from Millipore. Protease inhibitor mixture was from Roche. VprBP and non-targeting siRNAs were from Dharmacon. HDAC1 siRNA was from Qiagen. HDAC3 siRNA was from Santa Cruz Biotechnology. SYBR Select Master Mix was from Life Technologies. The lentiviral vectors pWPI, pWPI-Flag-Vpr, pWPI-Flag-Q65R, pWPI-Flag-R80A, and their packaging plasmids pCMV-VSV-G and psPax2 were described previously (10Romani B. Shaykh-Baygloo N. Aghasadeghi M.R. Allahbakhshi E. HIV-1 Vpr protein enhances proteasomal degradation of MCM10 DNA replication factor through the Cul4-DDB1[VprBP] E3 ubiquitin ligase to induce G2/M cell cycle arrest.J. Biol. Chem. 2015; 290: 17380-17389Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). pWPI-Flag-Vpx was constructed by cloning of Vpx from simian immunodeficiency virus (clone pPBj 1.9) of sooty mangabeys into pWPI using a strategy previously described (10Romani B. Shaykh-Baygloo N. Aghasadeghi M.R. Allahbakhshi E. HIV-1 Vpr protein enhances proteasomal degradation of MCM10 DNA replication factor through the Cul4-DDB1[VprBP] E3 ubiquitin ligase to induce G2/M cell cycle arrest.J. Biol. Chem. 2015; 290: 17380-17389Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). To generate an expression vector for HIV-1 Vpr, the vpr gene of HIV Gag-iGFP was cloned into pcDNA3.1. pRK5-HA-Ubiquitin-WT, pcDNA3.1-HDAC1-Flag,and pcDNA3.1-HDAC3-Flag were obtained from Addgene. pNL(AD8), pNL4–3-deltaE-EGFP, and pNL4.3.Luc.Env- were obtained from the NIH AIDS Research and Reference Reagent Program. The R5-tropic clone of HIV-1, pNL4.3.AD8. IRES_GFP_Nef-, was generated by cutting a 1.7-kbp fragment between the Kpnl and Bsml site in the envelope coding region from the R5-tropic clone pNL(AD8) and replacing this fragment for the corresponding region of pNL4.3_IRES_GFP_Nef-. The ΔVpr, Q65R, and R80A mutants were generated in the viral constructs using site-directed mutagenesis. All viral and lentiviral vectors were produced in HEK293T cells using the standard calcium phosphate transfection method. Viral and lentiviral particles were collected 48 h and 72 h post-transfection by ultracentrifugation at 35000 rpm for 2 h. Briefly, lentiviral vectors were produced by cotransfection of pWPI, pWPI-Flag-Vpr, pWPI-Flag-Vpr(Q65R), pWPI-Flag-Vpr(R80A), and pWPI-Flag-Vpx and the packaging plasmids, pCMV-VSV-G and psPax2. Lentiviral vectors were titrated in HEK293T cells using GFP signal. The VSV-G-pseudotyped pNL4.3.Luc.Env-(WT/ΔVpr) and pNL4–3-deltaE-EGFP (WT/ΔVpr/Q65R/R80A) were produced by cotransfection of the proviral plasmids with pCMV-VSV-G. pNL4.3.AD8. IRES_GFP_Nef- and its mutants (ΔVpr/Q65R/R80A) were produced by transfection of the proviral plasmids in HEK293T cells. pNL4.3.Luc.Env-(WT/ΔVpr) and pNL4.3.AD8. IRES_GFP_Nef- derived viruses were titrated in TZM-bl cells. p24 of the concentrated viral stock for pNL4–3-deltaE-EGFP (WT/ΔVpr/Q65R/R80A) was titrated using ELISA. HeLa cells were transduced with lentiviral vectors at an MOI of 2.0. Primary MDMs were infected at an MOI of 1.0 with fully replicative viruses or with 1 ng of p24 per 105 cells when infecting with single cycle VSV-G pseudotyped viruses. VprBP, HDAC1, HDAC3 and nontargeting siRNAs were transfected into inducible HeLa cell lines and/or primary MDMs using Lipofectamine RNAiMax Reagent according to the manufacturer's instructions. In order to fractionate HeLa and inducible HeLa cell lines, cells were lysed in 1 ml of 0.5% Triton lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 0.5% Triton, and protease inhibitor mixture). Cells were incubated for 10 min with mild agitation at 4 °C and then centrifuged at 6000 rpm for 10 min at 4 °C to pellet chromatin and other large insoluble debris. Supernatant was collected as the soluble fraction, and the pellet was resuspended in 2 ml of Benzonase buffer (50 mm Tris pH 8, 150 mm NaCl, 1.5 mm MgCl2, 0.1 mg/ml BSA, and protease inhibitor mixture). The resuspended pellet was centrifuged and supernatant was discarded. 1 μl of Benzonase enzyme (25 units/μl) was added to 1 ml of Benzonase buffer and this was used to resuspend the pellet and incubate it on ice for 60 min. The Benzonase-treated pellet was centrifuged at 13000 rpm for 10 min at 4 °C. Supernatant was collected as the fraction containing chromatin-bound proteins. Purity of the fractions was confirmed by the presence of GAPDH in soluble protein fraction and histone H3 in chromatin-bound protein fraction. Anti-Flag and anti-HA immunoprecipitations were performed using 40 μl of commercial antibodies conjugated to agarose beads. For anti-VprBP, HDAC1, HDAC2, HDAC3, and HDAC8 immunoprecipitations, antibodies (2 μg per immunoprecipitation) were incubated overnight at 4 °C with 50 μl of protein A-Sepharose beads in 1 ml of PBS supplemented with 5% FBS. All the immunoprecipitations were performed in the presence of 150 mm NaCl and 0.5% Triton X-100 for 2 h at 4 °C. For VprBP pull-down, 25 μg/ml ethidium bromide was also added to the solution to avoid the nonspecific pull-down of chromatin-bound proteins through DNA bridges. After thorough washes in 0.5% Triton lysis buffer, the anti-Flag immunoprecipitated proteins were eluted by adding 100 μg/ml Flag peptides. Immunoprecipitations using protein A-Sepharose beads and monoclonal anti-HA-agarose beads were released by treating the beads with 0.1 m glycine, pH 2.0, for 10 min on ice. Immunoprecipitated proteins and cellular fractions (30 μg) were resuspended in Laemmli buffer, heat-denatured for 5 min, and separated on 12% SDS-PAGE gels. Western blots were performed as described previously (10Romani B. Shaykh-Baygloo N. Aghasadeghi M.R. Allahbakhshi E. HIV-1 Vpr protein enhances proteasomal degradation of MCM10 DNA replication factor through the Cul4-DDB1[VprBP] E3 ubiquitin ligase to induce G2/M cell cycle arrest.J. Biol. Chem. 2015; 290: 17380-17389Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Supernatants of the infected cells were collected, inactivated with Triton X-100 (1% final concentration), and stored at −80 °C until analyzed. Levels of HIV-1 p24 were determined using HIV-1 p24 Antigen Capture assay (ABL Inc.) according to the manufacturer's instructions. Primary MDMs were infected at an MOI of 1.0 with single cycle VSV-G pseudotyped pNL4.3.Luc.Env-(WT/ΔVpr) that expresses luciferase in the infected cells. Seven days after infection, cells were washed with PBS and lysed directly in the tissue culture plates using Triton X-100 (1% final concentration). Luciferase was measured in the cell lysates using luciferase assay system (Promega). To analyze enrichment of class I HDACs and acetylated histones on the HIV-1 LTR, 5 × 105 MDMs were infected with pNL4.3.AD8. IRES_GFP_Nef- (WT and ΔVpr) at an MOI of 1.0. After 7 days, cells were looked under a fluorescent microscope for GFP-positive infected cells. If percentage of the infected cells was higher than 5%, we proceeded with the experiment. Cells were then fixed with 0.5% formaldehyde. After 5 min, fixation was quenched by adding glycine to a final concentration of 125 mm for 5 min and cells were then washed with PBS and harvested. Cells were lysed in TpA (0.25% Triton, 10 mm Tris pH8, 10 mm EDTA, 0.5 mm EGTA, protease inhibitor) for 5 min on ice and centrifuged. This would disrupt the cellular membrane and release the non-integrated proviruses. Cell nuclei were then washed with TpB (200 mm NaCl, 10 mm Tris, pH 8, 1 mm EDTA, 0.5 mm EGTA, protease inhibitor) and lysed in TpS (0.5% SDS, 0.5% Triton, 10 mm Tris pH8, 140 mm NaCl, 1 mm EDTA, 0.5 mm EGTA, protease inhibitor). Chromatin was sheared using Bioruptor XL sonicator (Diagenode) to obtain DNA fragments ranging from 200 to 800 bps. For each immunoprecipitation, 2 μg of antibody was conjugated to 10 μl of protein A and protein G Dynabeads (1:1 ratio) by overnight incubation of the beads/antibody in 500 μl of ChIP dilution buffer (1% Triton, 10 mm Tris, pH 8, 150 mm NaCl, 2 mm EDTA) at 4 °C. ChIPs were performed by adding the sonicated DNA to the antibody-conjugated Dynabeads and incubation at 4 °C for 4 h. Following extensive washes, the DNA of ChIP samples and nonimmunoprecipitated chromatin (input) were decross-linked in TpE (0.3% SDS, 50 mm Tris, pH 8, 10 mm EDTA, 0.4 m NaCl) by overnight incubation at 65 °C and extracted using the standard phenol-chloroform method. The DNA was then subjected to quantitative PCR. Occupancy of HIV-1 genome by class I HDACs and acetylated histones was analyzed using quantitative PCR. Briefly, the DNA that was pulled down using ChIP and the corresponding input DNA was subjected to PCR using the following primers: nuc-0-F 5′-CCACACACAAGGCTACTTCCCT-3′, nuc-0-R 5′-CAACTGGTACTAACTTGAAGCA-3′, nuc-1-F 5′-GTCTCTCTGGTTAGACCAGA-3′, nuc-1-R 5′-TACTTTGAGCACTCAAGGCA-3′, nuc-2-F 5′-AAAAATTTTGACTAGCGGAGGCT-3′, nuc-2-R 5′-CTTAACCGAATTTTTTCCCA-3′, ChIP-GAPDH-F 5′-CCTTCCCCTAGTCCCCAGAA-3′, ChIP-GAPDH-R 5′-AGCGCGAAAGGAAAGAAAGC-3′. A pair of primers were also designed against an intergenic region (gene accession number: AF254641.1), where no hyperacetylation of histones is expected, as follow: INT-F 5′-GTAGAGGAAGCGATCTGGGA-3′, INT-R 5′-CAAGGCCACTCTCGGCCTCT-3′. Amplification of the target sequences was detected using SYBR Select Master Mix, and the CT values were normalized to the inputs and the intergenic region. To assess whether Vpr affects class I HDACs at transcription level, HeLa-iFlag-Vpr and HeLa-iMock were induced by adding 1 μg/ml doxycycline. After 30 h, cells were harvested, and total RNA was extracted from 106 cells using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Using SuperScript II Reverse Transcriptase (Invitrogen) and oligo(dT)12–18 Primers (Invitrogen), RNA was reverse transcribed into cDNA. The cDNA was diluted 100-fold with DNase-free water. Quantitative PCR was performed on the diluted cDNA using SYBR Select Master Mix. Primers used in qPCR include: HDAC1-F 5′-TACGACGGGGATGTTGGAAA-3′, HDAC1-R 5′-ATTGGCTTTGTGAGGGCGAT-3′, HDAC2-F 5′-CCATGGCGTACAGTCAAGGA-3′, HDAC2-R 5′-TCATTTCTTCGGCAGTGGCT-3′, HDAC3-F 5′-TCTTTGAGTTCTGCTCGCGT-3′, HDAC3-R 5′-GCCAGAGGCCTCAAACTTCT-3′, HDAC8-F 5′-TCTCCAGAAGGTCAGCCAAGA-3′, HDAC8-R 5′-TCTTTGCATGATGCCACCCT-3′, GAPDH-F 5′-GAGAAGGCTGGGGCTCATTT-3′, GAPDH-R 5′-GCAGTGATGGCATGGACTGT-3′. Data were normalized to GAPDH and calculated using the 2−(ΔΔCT) method. To analyze expression levels of HIV-1 mRNA in infected MDMs, total mRNA was extracted using Ultrasens Viral RNA kit (Qiagen) and reverse transcribed using SuperScript II Reverse Transcriptase and oligo(dT)12–18 Primers. cDNA was then subjected to quantitative PCR using nuc-2-F and nuc-2-R primers. To compare integration of HIV-1 provirus in the presence or absence of Vpr, genome of infected MDMs was analyzed using Alu-PCR. Briefly, primary MDMs were infected with the single cycle VSV-G pseudotyped pNL4.3Env-.IRES.GFP WT and ΔVpr viruses. Four days post-infection, GFP-positive and -negative cells were sorted and the genomic DNA was extracted from 20,000 sorted and unsorted cells using DNeasy Blood & Tissue Kit (QIAGen). DNA was amplified using the forward primer Alu-F 5′-GCCTCCCAAAGTGCTGGGATTACAG-3′ that binds human Alu gene and the reverse primer HIV-LTR-outer-R 5′-TGCTAGAGATTTTCCACACTGA-3′ that binds HIV-1 LTR. One microliter of the amplified DNA was used for qPCR using HIV-LTR-F 5′-CTGGCTAACTAGGGAACCCACT-3′ and HIV-LTR-inner-R 5′-CTCAATAAAGCTTGCCTTGAGTGCTC-3′ primers. To amplify the total HIV-1 provirus (integrated and non-integrated), DNA was amplified using HIV-LTR-F and HIV-LTR-inner-R. Amplification of a specific region on HIV-1 LTR was detected using the probe LTR-Probe 5′-(FAM)-CTCAATAAAGCTTGCCTTGAGTGCTC-(TAMRA)-3′. Results of Alu-PCR were normalized to the total HIV-1 provirus. To analyze the cell cycle profile of inducible HeLa cell lines, they were induced by adding doxycycline and after 30 h their DNA content was labeled by propidium iodide staining and cells were analyzed as previously described (10Romani B. Shaykh-Baygloo N. Aghasadeghi M.R. Allahbakhshi E. HIV-1 Vpr protein enhances proteasomal degradation of MCM10 DNA replication factor through the Cul4-DDB1[VprBP] E3 ubiquitin ligase to induce G2/M cell cycle arrest.J. Biol. Chem. 2015; 290: 17380-17389Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). To examine purity and differentiation of MDMs, their surface markers were labeled using CD14 Mouse Anti-Human mAb, PE-Texas Red conjugate and APC/Cy7 anti-human CD16 for 30 min on ice. Cells were washed three times and analyzed using flow cytometry. To analyze infectivity of the single cycle HIV-1 in MDMs, they were infected with VSV-G-pseudotyped NL4–3-deltaE-EGFP (WT, ΔVpr, Q65R, and R80A). Four days post-infection, MDMs were analyzed for expression of GFP. All the analyses were performed using CyAn ADP Analyzer. To analyze acute versus latent infections, primary MDMs were infected with single cycle VSV-G pseudotyped GFP reporter WT/ΔVpr viruses for 4 days. The GFP-positive and -negative cells were sorted in PBS using an Influx cell sorter (BD Biosciences) and analyzed using Alu-PCR or Western blot. To analyze viral expression in macrophages, primary MDMs were infected with GFP reporter WT/ΔVpr viruses for 7 days. The GFP-positive cells were then sorted using an Influx cell sorter (BD Biosciences) and analyzed using Western blot. Student's t test or Analysis of variance (ANOVA) with Bonferroni's multiple comparison test was used for statistical analysis using GraphPad Prism 6.0. A value of p < 0.05 was considered statistically significant. Results were expressed as mean ± S.E.M. or mean ± S.D., and represent data from a minimum of three independent experiments unless otherwise stated. Since VprBP was reported to interact with HDAC1 on chromatin, we developed a fractionation method to examine the impact of Vpr on this interaction in a localized manner. Our fractions included soluble and chromatin-bound protein fractions, in which the soluble fraction contained both cytoplasmic proteins and soluble nuclear proteins, while the chromatin-bound protein fraction contained nuclear proteins released by treatment of the chromatin pellet with Benzonase DNase. A doxycycline-inducible cell line, HeLa-iFlag-Vpr cells, was used as previously described (10Romani B. Shaykh-Baygloo N. Aghasadeghi M.R. Allahbakhshi E. HIV-1 Vpr protein enhances proteasomal degradation of MCM10 DNA replication factor through the Cul4-DDB1[VprBP] E3 ubiquitin ligase to induce G2/M cell cycle arrest.J. Biol. Chem. 2015; 290: 17380-17389Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Expression of Flag-tagged HIV-1 Vpr was induced by adding doxycycline to HeLa-iFlag-Vpr cells and the quantities of class I HDACs were monitored in a time course experiment (Fig. 1A). A significant depletion of HDAC1 was found after 10 h in chromatin fraction followed by its depletion in the soluble fraction only after 30 h. Depletion of HDAC2 was only found significant in chromatin fraction 30 h post-induction with no significant depletion in the soluble fraction. Depletion of HDAC3 was initiated as early as 5 h post-induction in the chromatin fraction and then followed by its depletion in the soluble fraction 20 h post-induction. Depletion of HDAC8, which was only found in chromatin fraction, appeared significant after 20 h. Our results indicated that depletion of class I HDACs was more drastic on chromatin as the chromatin associated class I HDACs were depleted before the soluble class I HDACs. Chromatin depletion of HDAC3 was found to occur before other HDACs, suggesting more sensitivity of HDAC3 to expression of Vpr while HDAC2 was found the least sensitive member of class I HDACs. Expression of Vpr did not seem to affect the mRNA levels of class I HDACs (Fig. 1B) suggesting the down-regulation of HDACs occurs at protein level. Furthermore, we tested the effect of Vpr on other classes of HDACs (Fig. 1C). Expression of Vpr did not affect the protein levels of other classes of HDACs. Vpr is well-documented to induce a cell cycle arrest at G2/M phase through activation of ATR pathway (24Lai M. Zimmerman E.S. Planelles V. Chen J. Activation of the ATR pathway by human immunodeficiency virus type 1 Vpr involves its direct binding to chromatin in vivo.J. Virol. 2005; 79: 15443-15451Crossref PubMed Scopus (76) Google Scholar). The cell cycle arrest could potentially change protein contents of the cells. To examine whether the depletion of class I HDACs is a by-product of G2/M cell" @default.
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• W2216889613 title "HIV-1 Vpr Protein Induces Proteasomal Degradation of Chromatin-associated Class I HDACs to Overcome Latent Infection of Macrophages" @default.
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