Dynamic Modulation of HIV-1 Integrase Structure and Function by Cellular Lens Epithelium-derived Growth Factor (LEDGF) Protein
2008; Elsevier BV; Volume: 283; Issue: 46 Linguagem: Inglês
10.1074/jbc.m805843200
ISSN1083-351X
AutoresChristopher J. McKee, Jacques J. Kessl, Nikolozi Shkriabai, Mohd Jamal Dar, Alan Engelman, Mamuka Kvaratskhelia,
Tópico(s)Biochemical and Molecular Research
ResumoThe mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra- and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target. The mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra- and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target. Integration of the reverse-transcribed RNA genome into a host chromosome is an obligatory step for HIV-1 3The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; D, donor; FS, full-site; HS, half-site; IBD, integrase-binding domain; IN, integrase; mt, mutant; NTA, nitrilotriacetic acid; NTD, N-terminal domain; CCD, catalytic core domain; CTD, C-terminal domain; LEDGF, lens epithelium-derived growth factor; NHS, N-hydroxysuccinimide; HPG, p-hydroxyphenylglyoxal; S, substrate; P, product; PIC, preintegration complex; Di, dimer; Tet, tetramer; M, modifier; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. replication (reviewed in Ref. 1Brown P.O. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory, Plainview, NY1997: 161-204Google Scholar). This process is catalyzed by the retroviral enzyme integrase (IN) in two reaction steps. In the first step, which is called 3′-processing and takes place shortly after the cDNA is made, IN hydrolyzes a GT dinucleotide from each end of the viral DNA. In the second step, IN catalyzes concerted integration of the processed viral DNA ends into chromosomal DNA. The sites of attack on the two target DNA strands are separated by 5 bp, which leads to dissociation of the small double-stranded DNA fragment between the attachment sites. The subsequent repair of the intermediate species by cellular enzymes completes the integration reaction. HIV-1 IN consists of three distinct structural and functional domains. The N-terminal domain (NTD) (residues 1–50) contains conserved pairs of histidine and cysteine residues that bind zinc (2Bushman F.D. Engelman A. Palmer I. Wingfield P. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3428-3432Crossref PubMed Scopus (334) Google Scholar, 3Cai M. Zheng R. Caffrey M. Craigie R. Clore G.M. Gronenborn A.M. Nat. Struct. Biol. 1997; 4: 567-577Crossref PubMed Scopus (307) Google Scholar), which contributes to IN multimerization and its catalytic function (4Zheng R. Jenkins T.M. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13659-13664Crossref PubMed Scopus (311) Google Scholar, 5Lee S.P. Xiao J. Knutson J.R. Lewis M.S. Han M.K. Biochemistry. 1997; 36: 173-180Crossref PubMed Scopus (158) Google Scholar). The catalytic core domain (CCD) (residues 51–212) contains three acidic residues, Asp-64, Asp-116, and Glu-152, which play a key role in coordinating active site divalent metal ions (6Dyda F. Hickman A.B. Jenkins T.M. Engelman A. Craigie R. Davies D.R. Science. 1994; 266: 1981-1986Crossref PubMed Scopus (730) Google Scholar, 7Goldgur Y. Dyda F. Hickman A.B. Jenkins T.M. Craigie R. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9150-9154Crossref PubMed Scopus (371) Google Scholar). The C-terminal domain (CTD) (residues 213–288) also contributes to functional IN multimerization (8Andrake M.D. Skalka A.M. J. Biol. Chem. 1995; 270: 29299-29306Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 9Jenkins T.M. Engelman A. Ghirlando R. Craigie R. J. Biol. Chem. 1996; 271: 7712-7718Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Results of structural biology studies revealed each individual domain as a dimer (3Cai M. Zheng R. Caffrey M. Craigie R. Clore G.M. Gronenborn A.M. Nat. Struct. Biol. 1997; 4: 567-577Crossref PubMed Scopus (307) Google Scholar, 6Dyda F. Hickman A.B. Jenkins T.M. Engelman A. Craigie R. Davies D.R. Science. 1994; 266: 1981-1986Crossref PubMed Scopus (730) Google Scholar, 7Goldgur Y. Dyda F. Hickman A.B. Jenkins T.M. Craigie R. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9150-9154Crossref PubMed Scopus (371) Google Scholar, 10Eijkelenboom A.P. Lutzke R.A. Boelens R. Plasterk R.H. Kaptein R. Hard K. Nat. Struct. Biol. 1995; 2: 807-810Crossref PubMed Scopus (220) Google Scholar, 11Lodi P.J. Ernst J.A. Kuszewski J. Hickman A.B. Engelman A. Craigie R. Clore G.M. Gronenborn A.M. Biochemistry. 1995; 34: 9826-9833Crossref PubMed Scopus (272) Google Scholar) and more recent two-domain crystal structures comprised of the CCD and CTD (12Chen J.C. Krucinski J. Miercke L.J. Finer-Moore J.S. Tang A.H. Leavitt A.D. Stroud R.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8233-8238Crossref PubMed Scopus (383) Google Scholar) or NTD and CCD (13Wang J.Y. Ling H. Yang W. Craigie R. EMBO J. 2001; 20: 7333-7343Crossref PubMed Scopus (313) Google Scholar) likewise unveiled dimeric organizations. Functional studies suggested that a dimer of full-length IN could suffice to process each 3′ end, whereas a tetramer is required to integrate both viral DNA ends into chromosomal DNA (14Faure A. Calmels C. Desjobert C. Castroviejo M. Caumont-Sarcos A. Tarrago-Litvak L. Litvak S. Parissi V. Nucleic Acids Res. 2005; 33: 977-986Crossref PubMed Scopus (168) Google Scholar, 15Guiot E. Carayon K. Delelis O. Simon F. Tauc P. Zubin E. Gottikh M. Mouscadet J.F. Brochon J.C. Deprez E. J. Biol. Chem. 2006; 281: 22707-22719Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 16Li M. Mizuuchi M. Burke Jr., T.R. Craigie R. EMBO J. 2006; 25: 1295-1304Crossref PubMed Scopus (209) Google Scholar). Efforts to determine the complete IN structure have been impeded by limited protein solubility and/or the inherent flexibility of the three-domain enzyme. Full-length IN interestingly exists as a mixture of monomers, dimers, tetramers, and higher order species in the absence of DNA (9Jenkins T.M. Engelman A. Ghirlando R. Craigie R. J. Biol. Chem. 1996; 271: 7712-7718Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 17van Gent D.C. Elgersma Y. Bolk M.W. Vink C. Plasterk R.H. Nucleic Acids Res. 1991; 19: 3821-3827Crossref PubMed Scopus (99) Google Scholar, 18Vincent K.A. Ellison V. Chow S.A. Brown P.O. J. Virol. 1993; 67: 425-437Crossref PubMed Google Scholar, 19Deprez E. Tauc P. Leh H. Mouscadet J.F. Auclair C. Brochon J.C. Biochemistry. 2000; 39: 9275-9284Crossref PubMed Scopus (131) Google Scholar). While in vitro analysis with model DNA substrates demonstrated that IN alone could catalyze 3′-processing and DNA strand transfer reactions, the in vivo function of the enzyme is likely to be regulated by a number of viral and cellular proteins. Following the completion of reverse transcription, the newly synthesized cDNA remains associated with several viral proteins and recruits host factors to form the preintegration complex (PIC) (20Bukrinsky M.I. Sharova N. McDonald T.L. Pushkarskaya T. Tarpley W.G. Stevenson M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6125-6129Crossref PubMed Scopus (393) Google Scholar, 21Farnet C.M. Bushman F.D. Cell. 1997; 88: 483-492Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 22Miller M.D. Farnet C.M. Bushman F.D. J. Virol. 1997; 71: 5382-5390Crossref PubMed Google Scholar, 23Li L. Olvera J.M. Yoder K.E. Mitchell R.S. Butler S.L. Lieber M. Martin S.L. Bushman F.D. EMBO J. 2001; 20: 3272-3281Crossref PubMed Scopus (299) Google Scholar, 24Lin C.W. Engelman A. J. Virol. 2003; 77: 5030-5036Crossref PubMed Scopus (121) Google Scholar, 25Llano M. Delgado S. Vanegas M. Poeschla E.M. J. Biol. Chem. 2004; 279: 55570-55577Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 26Iordanskiy S. Berro R. Altieri M. Kashanchi F. Bukrinsky M. Retrovirology. 2006; 3: 4Crossref PubMed Scopus (76) Google Scholar, 27Cherepanov P. Maertens G. Proost P. Devreese B. Van Beeumen J. Engelborghs Y. De Clercq E. Debyser Z. J. Biol. Chem. 2003; 278: 372-381Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar, 28Maertens G. Cherepanov P. Pluymers W. Busschots K. De Clercq E. Debyser Z. Engelborghs Y. J. Biol. Chem. 2003; 278: 33528-33539Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 29Turlure F. Devroe E. Silver P.A. Engelman A. Front. Biosci. 2004; 9: 3187-3208Crossref PubMed Scopus (141) Google Scholar). Of these, transcriptional co-activator p75, also known as lens epithelium-derived growth factor (LEDGF), is the principal cellular interactor of HIV-1 IN (27Cherepanov P. Maertens G. Proost P. Devreese B. Van Beeumen J. Engelborghs Y. De Clercq E. Debyser Z. J. Biol. Chem. 2003; 278: 372-381Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar, 28Maertens G. Cherepanov P. Pluymers W. Busschots K. De Clercq E. Debyser Z. Engelborghs Y. J. Biol. Chem. 2003; 278: 33528-33539Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 29Turlure F. Devroe E. Silver P.A. Engelman A. Front. Biosci. 2004; 9: 3187-3208Crossref PubMed Scopus (141) Google Scholar). A number of recent studies have indicated that LEDGF is critically important for effective HIV-1 integration and viral replication (30De Rijck J. Vandekerckhove L. Gijsbers R. Hombrouck A. Hendrix J. Vercammen J. Engelborghs Y. Christ F. Debyser Z. J. Virol. 2006; 80: 11498-11509Crossref PubMed Scopus (151) Google Scholar, 31Vandekerckhove L. Christ F. Van Maele B. De Rijck J. Gijsbers R. Van den Haute C. Witvrouw M. Debyser Z. J. Virol. 2006; 80: 1886-1896Crossref PubMed Scopus (184) Google Scholar, 32Hombrouck A. De Rijck J. Hendrix J. Vandekerckhove L. Voet A. De Maeyer M. Witvrouw M. Engelborghs Y. Christ F. Gijsbers R. Debyser Z. PLoS. Pathog. 2007; 3: e47Crossref PubMed Scopus (105) Google Scholar, 33Llano M. Saenz D.T. Meehan A. Wongthida P. Peretz M. Walker W.H. Teo W. Poeschla E.M. Science. 2006; 314: 461-464Crossref PubMed Scopus (431) Google Scholar). RNA interference (RNAi)-mediated knock-down of endogenous LEDGF to below detectable levels resulted in reduction of infection to 3.5% of that observed in the presence of normal cells (33Llano M. Saenz D.T. Meehan A. Wongthida P. Peretz M. Walker W.H. Teo W. Poeschla E.M. Science. 2006; 314: 461-464Crossref PubMed Scopus (431) Google Scholar). Similarly significantly reduced levels of HIV-1 infection were detected in LEDGF knock-out mouse embryo fibroblasts (34Shun M.C. Raghavendra N.K. Vandegraaff N. Daigle J.E. Hughes S. Kellam P. Cherepanov P. Engelman A. Genes Dev. 2007; 21: 1767-1778Crossref PubMed Scopus (384) Google Scholar, 35Marshall H.M. Ronen K. Berry C. Llano M. Sutherland H. Saenz D. Bickmore W. Poeschla E. Bushman F.D. PLoS ONE. 2007; 2: e1340Crossref PubMed Scopus (199) Google Scholar). Expression of recombinant HIV-1 IN in human cells revealed that LEDGF protects the viral protein from proteasomal degradation and tethers it to chromosomal DNA (25Llano M. Delgado S. Vanegas M. Poeschla E.M. J. Biol. Chem. 2004; 279: 55570-55577Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 28Maertens G. Cherepanov P. Pluymers W. Busschots K. De Clercq E. Debyser Z. Engelborghs Y. J. Biol. Chem. 2003; 278: 33528-33539Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 36Emiliani S. Mousnier A. Busschots K. Maroun M. Van Maele B. Tempe D. Vandekerckhove L. Moisant F. Ben-Slama L. Witvrouw M. Christ F. Rain J.C. Dargemont C. Debyser Z. Benarous R. J. Biol. Chem. 2005; 280: 25517-25523Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 37Llano M. Vanegas M. Fregoso O. Saenz D. Chung S. Peretz M. Poeschla E.M. J. Virol. 2004; 78: 9524-9537Crossref PubMed Scopus (260) Google Scholar, 38Llano M. Vanegas M. Hutchins N. Thompson D. Delgado S. Poeschla E.M. J. Mol. Biol. 2006; 360: 760-773Crossref PubMed Scopus (160) Google Scholar). Accordingly, LEDGF primarily functions during HIV-1 infection to tether PICs to active genes during integration (34Shun M.C. Raghavendra N.K. Vandegraaff N. Daigle J.E. Hughes S. Kellam P. Cherepanov P. Engelman A. Genes Dev. 2007; 21: 1767-1778Crossref PubMed Scopus (384) Google Scholar). In vitro assays with purified recombinant proteins furthermore demonstrated that LEDGF binds directly to IN, which significantly stimulates its enzymatic activities (27Cherepanov P. Maertens G. Proost P. Devreese B. Van Beeumen J. Engelborghs Y. De Clercq E. Debyser Z. J. Biol. Chem. 2003; 278: 372-381Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar, 39Cherepanov P. Devroe E. Silver P.A. Engelman A. J. Biol. Chem. 2004; 279: 48883-48892Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 40Turlure F. Maertens G. Rahman S. Cherepanov P. Engelman A. Nucleic Acids Res. 2006; 34: 1653-1675Crossref PubMed Scopus (151) Google Scholar, 41Cherepanov P. Nucleic Acids Res. 2007; 35: 113-124Crossref PubMed Scopus (146) Google Scholar, 42Pandey K.K. Sinha S. Grandgenett D.P. J. Virol. 2007; 81: 3969-3979Crossref PubMed Scopus (53) Google Scholar, 43Raghavendra N.K. Engelman A. Virology. 2007; 360: 1-5Crossref PubMed Scopus (42) Google Scholar, 44Yu F. Jones G.S. Hung M. Wagner A.H. MacArthur H.L. Liu X. Leavitt S. McDermott M.J. Tsiang M. Biochemistry. 2007; 46: 2899-2908Crossref PubMed Scopus (28) Google Scholar). The N-terminal part of LEDGF contains a PWWP domain, nuclear localization signal, and dual copy of the AT-hook DNA binding motif (reviewed in Ref. 45Engelman A. Cherepanov P. PLoS. Pathog. 2008; 4: e1000046Crossref PubMed Scopus (192) Google Scholar and Fig. 1). These conserved elements primarily mediate LEDGF association with chromatin (38Llano M. Vanegas M. Hutchins N. Thompson D. Delgado S. Poeschla E.M. J. Mol. Biol. 2006; 360: 760-773Crossref PubMed Scopus (160) Google Scholar, 40Turlure F. Maertens G. Rahman S. Cherepanov P. Engelman A. Nucleic Acids Res. 2006; 34: 1653-1675Crossref PubMed Scopus (151) Google Scholar). An evolutionarily conserved domain in the C-terminal region (residues 347–429) mediates the interaction with IN and was thus termed the IN-binding domain (IBD) (39Cherepanov P. Devroe E. Silver P.A. Engelman A. J. Biol. Chem. 2004; 279: 48883-48892Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 46Vanegas M. Llano M. Delgado S. Thompson D. Peretz M. Poeschla E. J. Cell Sci. 2005; 118: 1733-1743Crossref PubMed Scopus (147) Google Scholar). The solution structure of the LEDGF IBD and its co-crystallization with the IN CCD has been recently reported (47Cherepanov P. Sun Z.Y. Rahman S. Maertens G. Wagner G. Engelman A. Nat. Struct. Mol. Biol. 2005; 12: 526-532Crossref PubMed Scopus (205) Google Scholar, 48Cherepanov P. Ambrosio A.L. Rahman S. Ellenberger T. Engelman A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17308-17313Crossref PubMed Scopus (356) Google Scholar). Interestingly, the IBD docks into a relatively small cavity at the CCD dimer interface, contacting both IN subunits (48Cherepanov P. Ambrosio A.L. Rahman S. Ellenberger T. Engelman A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17308-17313Crossref PubMed Scopus (356) Google Scholar). The importance of the interacting amino acids revealed from the crystal structure has been validated by site directed mutagenesis in the context of full-length recombinant proteins (29Turlure F. Devroe E. Silver P.A. Engelman A. Front. Biosci. 2004; 9: 3187-3208Crossref PubMed Scopus (141) Google Scholar, 36Emiliani S. Mousnier A. Busschots K. Maroun M. Van Maele B. Tempe D. Vandekerckhove L. Moisant F. Ben-Slama L. Witvrouw M. Christ F. Rain J.C. Dargemont C. Debyser Z. Benarous R. J. Biol. Chem. 2005; 280: 25517-25523Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 47Cherepanov P. Sun Z.Y. Rahman S. Maertens G. Wagner G. Engelman A. Nat. Struct. Mol. Biol. 2005; 12: 526-532Crossref PubMed Scopus (205) Google Scholar, 49Busschots K. Voet A. De Maeyer M. Rain J.C. Emiliani S. Benarous R. Desender L. Debyser Z. Christ F. J. Mol. Biol. 2007; 365: 1480-1492Crossref PubMed Scopus (118) Google Scholar, 50Rahman S. Lu R. Vandegraaff N. Cherepanov P. Engelman A. Virology. 2007; 357: 79-90Crossref PubMed Scopus (61) Google Scholar) and by the out-growth of resistant viral strains in the presence of a dominant-interfering LEDGF fragment (32Hombrouck A. De Rijck J. Hendrix J. Vandekerckhove L. Voet A. De Maeyer M. Witvrouw M. Engelborghs Y. Christ F. Gijsbers R. Debyser Z. PLoS. Pathog. 2007; 3: e47Crossref PubMed Scopus (105) Google Scholar). However, mutagenesis studies have also indicated that full-length IN-LEDGF interactions extend beyond the contacts observed in the co-crystal structure of the isolated domains (28Maertens G. Cherepanov P. Pluymers W. Busschots K. De Clercq E. Debyser Z. Engelborghs Y. J. Biol. Chem. 2003; 278: 33528-33539Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). We have undertaken a number of innovative biochemical approaches to characterize the structural and mechanistic foundations between the full-length interacting partners, which has revealed a highly dynamic nature for the interactions between free IN subunits. LEDGF moreover strongly stabilized the IN subunit-subunit contacts. Mass spectrometric surface topology studies furthermore uncovered novel protein-protein contacts, which lie outside of the central IBD-CCD co-crystal structure. Mutational analysis confirmed the importance of the identified residues and indicated a strong correlation between IN tetramer formation and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure/function, and highlight the potential to exploit the highly dynamic nature of IN subunit interactions as a novel therapeutic target. Expression Plasmids and Recombinant Proteins—HIV-1 IN proteins were expressed from pKBIN6Hthr, which was derived from pKB-IN6H (28Maertens G. Cherepanov P. Pluymers W. Busschots K. De Clercq E. Debyser Z. Engelborghs Y. J. Biol. Chem. 2003; 278: 33528-33539Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar) by replacing amino acids VDKLAAALE upstream from the C-terminal His6 affinity tag with LVPRGSALE (thrombin cleavage site underlined) by PCR-directed mutagenesis. Mutations were also introduced into pKBIN6Hthr using PCR, and the coding regions of plasmids created via PCR were verified by DNA sequencing. Wild-type and mutant IN proteins were purified according to the previously described procedure (41Cherepanov P. Nucleic Acids Res. 2007; 35: 113-124Crossref PubMed Scopus (146) Google Scholar). Purified recombinant LEDGF, mutant (mt) LEDGF, and IBD (Fig. 1) were obtained as described previously (28Maertens G. Cherepanov P. Pluymers W. Busschots K. De Clercq E. Debyser Z. Engelborghs Y. J. Biol. Chem. 2003; 278: 33528-33539Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 39Cherepanov P. Devroe E. Silver P.A. Engelman A. J. Biol. Chem. 2004; 279: 48883-48892Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 47Cherepanov P. Sun Z.Y. Rahman S. Maertens G. Wagner G. Engelman A. Nat. Struct. Mol. Biol. 2005; 12: 526-532Crossref PubMed Scopus (205) Google Scholar). IN 3′-Processing and DNA Strand Transfer Activities—The 32P-labeled 21-mer synthetic double-stranded DNA (50 nm) mimicking the U5 viral end sequence was used as substrate. The concentrations of wild type and mutant IN proteins as well as LEDGF and LEDGF IBD included in the reactions are indicated in the figure legends. The reactions were carried out at 37 °C for 1 h in buffer containing: 50 mm MOPS (pH 7.2), 2 mm β-mercaptoethanol, 10 mm MnCl2, 1 mm CHAPS, 50 mm NaCl, and stopped with 50 mm EDTA. Reaction products were subjected to denaturing polyacrylamide gel electrophoresis and visualized using a Storm 860 Phosphorimager (Amersham Biosciences). Concerted Integration Assay—These assays were performed as described previously (43Raghavendra N.K. Engelman A. Virology. 2007; 360: 1-5Crossref PubMed Scopus (42) Google Scholar). Briefly, the 972 bp ScaI-DraIII restriction fragment from pU3U5 (51Cherepanov P. Surratt D. Toelen J. Pluymers W. Griffith J. De Clercq E. Debyser Z. Nucleic Acids Res. 1999; 27: 2202-2210Crossref PubMed Scopus (41) Google Scholar) served as donor DNA and was 5′-end-labeled with [32P]ATP and T4 polynucleotide kinase. HIV-1 IN (400 nm) was assembled with the labeled donor substrate (18 nm) in the presence of 20 mm HEPES (pH 7.0), 5 mm dithiothreitol, 10 mm MgCl2, 25 μm ZnCl2, 100 mm NaCl, 5% DMSO, 10% PEG 6000. Ligands (IBD or LEDGF) were added before preincubation for 20 min at room temperature. Reactions (25-μl final volume) were initiated by adding 500 ng of circular target DNA (pGEM, Promega), and the mixtures were incubated for 1 h at 37 °C. Reactions were stopped by adding 10 mm EDTA, 0.2% SDS, and 1 mg/ml proteinase K. After ethanol precipitation, samples were subjected to 0.6% agarose gel electrophoresis for 6 h at 50 V. The gels were dried, and the labeled DNA products were detected using the Storm 860 Phosphorimager. Subunit Exchange Assay—His-tagged IN (1 μm) was preincubated with or without ligand (2 μm LEDGF or 2 μm mtLEDGF) in exchange buffer (25 mm Hepes, pH 7.1, 200 mm NaCl, 4% glycerol, 2 mm β-mercaptoethanol) for 30 min at room temperature. Tag-free IN (1 μm) was then added and incubated for the indicated times. Aliquots were then briefly centrifuged 2 min at 1,000 × g, and supernatants were pulled-down by Ni-nitrilotriacetic acid (NTA) resin (GE Healthcare) for 10 min in the presence of bovine serum albumin (0.1 mg/ml). The IN-bound resin was then washed three times with buffer containing 50 mm HEPES (pH 7.1), 200 mm NaCl, 2 mm MgCl2, 100 mm imidazole, and 0.1% (v/v) Nonidet P40. The bound proteins were subjected to SDS-PAGE separation and visualized by Coomassie Blue stain. Mass Spectrometric Footprinting—In parallel reactions, free IN and IN+LEDGF were first incubated at room temperature for 30 min and then subjected to treatments at 37 °C with 1 mmN-hydroxysuccinimide (NHS)-biotin for 30 min or 20 mm p-hydroxyphenylglyoxal (HPG) for 60 min. These concentrations of modifying reagents were chosen because comparative pulldown experiments with untreated and modified IN-LEDGF complexes indicated that under these conditions the integrity of the preassembled protein-protein complex was fully preserved (data not shown). NHS-biotin treatment was carried out in buffer containing 50 mm HEPES (pH 8.0), 150 mm NaCl, 10 mm MgCl2. The HPG modifications were performed in 50 mm HEPES (pH 8.0), 50 mm boric acid, 150 mm NaCl. The reactions were quenched by excess Lys and Arg using free amino acid forms. IN-LEDGF complexes were selectively pulled-down using Ni-NTA resin. The bound proteins were separated by denaturing SDS-PAGE and visualized by Microwave Blue stain (Protiga, Gaithersburg, MD). IN bands were excised, destained, and subjected to in-gel proteolysis with 0.5 μg of trypsin. The tryptic peptides were analyzed with the Axima-CFR MALDI-ToF instrument (Shimadzu) using α-cyano-4-hydroxy-cinnamic acid as a matrix. Size Exclusion Chromatography—Experiments were performed with a Superdex 200 10/300 GL column (GE Healthcare) at 0.5 ml/min in buffer containing 50 mm HEPES (pH 7.4), 750 mm NaCl, and 10% glycerol. The column was calibrated with the following proteins: conalbumin (75,000 Da), carbonic anhydrase (29,000 Da), ribonuclease A (13,700 Da), and aprotinin (6,500 Da). Proteins were detected by absorbance at 280 nm. Molecular Modeling—The model of the NTD-CCD tetramer bound to the LEDGF IBD was generated by overlaying the CCDs within PDB structures 2B4J (48Cherepanov P. Ambrosio A.L. Rahman S. Ellenberger T. Engelman A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17308-17313Crossref PubMed Scopus (356) Google Scholar) and 1K6Y (13Wang J.Y. Ling H. Yang W. Craigie R. EMBO J. 2001; 20: 7333-7343Crossref PubMed Scopus (313) Google Scholar) using the Insight II software package (Accelrys Inc., San Diego) on a Silicon Graphics O2 work station. The constructed model was then energy-minimized by the same software package using the CFF91 force field and steepest descent method. LEDGF Binding Affinities to Wild-type and Mutant INs—LEDGF (50–650 nm) was incubated with 100 nm His-tagged IN (WT or mutant) in binding buffer (50 mm Hepes (pH 7.1), 200 mm NaCl, 2 mm MgCl2, 100 mm imidazole, 0.1% (v/v) Nonidet P40) for 60 min at room temperature. Samples were then briefly centrifuged for 2 min at 1,000 × g, and supernatants were pulled-down by Ni-NTA resin for 30 min in the presence of bovine serum albumin (0.1 mg/ml). The resin was then washed three times with the same buffer, and the bound proteins were separated by SDS-PAGE. LEDGF was detected by Western blot analysis using a mouse monoclonal LEDGF antibody (BD Biosciences) and quantified using Image software (NIH). Plotting and curve fitting was performed with Origin 8 software (OriginLab). Nonspecific signal was not detected when LEDGF was incubated with Ni-NTA beads in the absence of IN (data not shown). We previously reported that LEDGF significantly stimulated the in vitro activities of HIV-1 IN whereas the isolated IBD failed to do so (39Cherepanov P. Devroe E. Silver P.A. Engelman A. J. Biol. Chem. 2004; 279: 48883-48892Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 40Turlure F. Maertens G. Rahman S. Cherepanov P. Engelman A. Nucleic Acids Res. 2006; 34: 1653-1675Crossref PubMed Scopus (151) Google Scholar). As these assays utilized relatively long blunt-ended viral DNA substrates and DNA strand transfer product formation as read-out, we reanalyzed the effects of these two proteins on IN function using an oligonucleotide-based assay that monitors the formation of 3′-processing and DNA strand transfer reaction products on denaturing sequencing gels (Fig. 2) (52Bushman F.D. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1339-1343Crossref PubMed Scopus (367) Google Scholar). The results in panel B (lanes 1–6) revealed stimulation of IN DNA strand transfer activity by the LEDGF IBD under these assay conditions. It should be noted that in this setting the 19-mer 3′-processing reaction product is the substrate for the second catalytic step (Fig. 2A). To dissect if the IBD directly enhanced IN 3′ processing activity, a selective IN strand transfer inhibitor (53Zhang X. Pais G.C. Svarovskaia E.S. Marchand C. Johnson A.A. Karki R.G. Nicklaus M.C. Pathak V.K. Pommier Y. Burke T.R. Bioorg. Med. Chem. Lett. 2003; 13: 1215-1219Crossref PubMed Scopus (66) Google Scholar, 54Svarovskaia E.S. Barr R. Zhang X. Pais G.C. Marchand C. Pommier Y. Burke Jr., T.R. Pathak V.K. J. Virol. 2004; 78: 3210-3222Crossref PubMed Scopus (98) Google Scholar) was included in the experiment (supplemental Fig. S1). The 19-mer reaction product accumulated under these conditions, revealing significant stimulation of IN 3′ processing activity by the LEDGF IBD (supplemental Fig. S1). For control experiments we analyzed the D366N point mtLEDGF, which is defective for IN binding in vitro (47Cherepanov P. Sun Z.Y. Rahman S. Maertens G. Wagner G. Engelman A. Nat. Struct. Mol. Biol. 2005; 12: 526-532Crossref PubMed Scopus (205) Google Scholar) and in yeast cells (50Rahman S. Lu R. Vandegraaff N. Cherepanov P. Engelman A. Virology. 2007; 357: 79-90Crossref PubMed Scopus (61) Google Scholar),
Referência(s)