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- W2088335428 abstract "We have reported that human immunodeficiency virus type 1 (HIV-1) integrase (IN) forms a specific nuclear complex with human lens epithelium-derived growth factor/transcription co-activator p75 (LEDGF/p75) protein. We now studied the IN-LEDGF/p75 interaction and nuclear import of IN in living cells using fusions of IN and LEDGF/p75 with enhanced green fluorescent protein and far-red fluorescent protein HcRed1. We show that both the N-terminal zinc binding domain and the central core domains of IN are involved in the interaction with LEDGF/p75. Both domains are essential for nuclear localization of IN as well as for the association of IN with condensed chromosomes during mitosis. However, upon overexpression of LEDGF/p75, the core domain fragment of IN was recruited to the nuclei and mitotic chromosomes with a distribution pattern characteristic of the full-length protein, indicating that it harbors the main determinant for interaction with LEDGF/p75. Although the C-terminal domain of IN was dispensable for nuclear/chromosomal localization, a fusion of the C-terminal IN fragment with enhanced green fluorescent protein was found exclusively in the nucleus, with a diffuse nuclear/nucleolar distribution, suggesting that the C-terminal domain may also play a role in the nuclear import of IN. In contrast to LEDGF/p75, its alternative splice variant, p52, did not interact with HIV-1 IN in vitro and in living cells. Finally, RNA interference-mediated knock-down of endogenous LEDGF/p75 expression abolished nuclear/chromosomal localization of IN. We conclude, therefore, that the interaction with LEDGF/p75 accounts for the karyophilic properties and chromosomal targeting of HIV-1 IN. We have reported that human immunodeficiency virus type 1 (HIV-1) integrase (IN) forms a specific nuclear complex with human lens epithelium-derived growth factor/transcription co-activator p75 (LEDGF/p75) protein. We now studied the IN-LEDGF/p75 interaction and nuclear import of IN in living cells using fusions of IN and LEDGF/p75 with enhanced green fluorescent protein and far-red fluorescent protein HcRed1. We show that both the N-terminal zinc binding domain and the central core domains of IN are involved in the interaction with LEDGF/p75. Both domains are essential for nuclear localization of IN as well as for the association of IN with condensed chromosomes during mitosis. However, upon overexpression of LEDGF/p75, the core domain fragment of IN was recruited to the nuclei and mitotic chromosomes with a distribution pattern characteristic of the full-length protein, indicating that it harbors the main determinant for interaction with LEDGF/p75. Although the C-terminal domain of IN was dispensable for nuclear/chromosomal localization, a fusion of the C-terminal IN fragment with enhanced green fluorescent protein was found exclusively in the nucleus, with a diffuse nuclear/nucleolar distribution, suggesting that the C-terminal domain may also play a role in the nuclear import of IN. In contrast to LEDGF/p75, its alternative splice variant, p52, did not interact with HIV-1 IN in vitro and in living cells. Finally, RNA interference-mediated knock-down of endogenous LEDGF/p75 expression abolished nuclear/chromosomal localization of IN. We conclude, therefore, that the interaction with LEDGF/p75 accounts for the karyophilic properties and chromosomal targeting of HIV-1 IN. The human immunodeficiency virus (HIV), 1The abbreviations used are: HIV, human immunodeficiency virus; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Ct, C-terminal; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; IN, integrase; LEDGF, lens epithelium-derived growth factor/transcription co-activator p75; MA, matrix protein; NLS, nuclear localization signal; Nt, N-terminal; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline; PIC, preintegration complex; siRNA, short interfering RNA. the causative agent of AIDS, belongs to the Lentiviridae genus of retroviruses. The early steps of HIV replication include reverse transcription of the diploid viral RNA genome into a double-stranded linear DNA replica and integration into a host cell chromosome. Reverse transcription takes place in the cytoplasm of the infected cell and results in the formation of a compact and stable preintegration complex (PIC), containing the viral reverse-transcribed genome and a number of virion-derived and cellular proteins. HIV and other lentiviruses are able to productively infect non-dividing, terminally differentiated cells, a feature distinguishing them from oncoretroviruses, which require cell division for productive infection (1Gartner S. Markovits P. Markovitz D.M. Kaplan M.H. Gallo R.C. Popovic M. Science. 1986; 233: 215-219Crossref PubMed Scopus (1357) Google Scholar, 2Weinberg J.B. Matthews T.J. Cullen B.R. Malim M.H. J. Exp. Med. 1991; 174: 1477-1482Crossref PubMed Scopus (305) Google Scholar, 3Roe T. Reynolds T.C. Yu G. Brown P.O. EMBO J. 1993; 12: 2099-2108Crossref PubMed Scopus (798) Google Scholar, 4Lewis P.F. Emerman M. J. Virol. 1994; 68: 510-516Crossref PubMed Google Scholar). Previous work has characterized the nuclear import of HIV-1 PICs as an active, energy-dependent process (5Bukrinsky M.I. Sharova N. Dempsey M.P. Stanwick T.L. Bukrinskaya A.G. Haggerty S. Stevenson M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6580-6584Crossref PubMed Scopus (507) Google Scholar), yet its mechanism has remained a puzzle. The determinants of HIV nuclear import that have been suggested so far are: the nuclear localization signals (NLSs) of the viral matrix (MA), Vpr, and integrase (IN) proteins, and the central DNA flap (for reviews see Refs. 6Fouchier R.A. Malim M.H. Adv. Virus Res. 1999; 52: 275-299Crossref PubMed Google Scholar, 7Vodicka M.A. Somat. Cell Mol. Genet. 2001; 26: 35-49Crossref PubMed Scopus (29) Google Scholar, 8Sherman M.P. Greene W.C. Microbes Infect. 2002; 4: 67-73Crossref PubMed Scopus (104) Google Scholar). The latter has been reported to be essential for nuclear import of HIV PICs and viral replication (9Zennou V. Petit C. Guetard D. Nerhbass U. Montagnier L. Charneau P. Cell. 2000; 101: 173-185Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). Although the effect of the central DNA flap appears to be viral strain- and host cell-dependent (10Limon A. Nakajima N. Lu R. Ghory H.Z. Engelman A. J. Virol. 2002; 76: 12078-12086Crossref PubMed Scopus (88) Google Scholar, 11Dvorin J.D. Bell P. Maul G.G. Yamashita M. Emerman M. Malim M.H. J. Virol. 2002; 76: 12087-12096Crossref PubMed Scopus (144) Google Scholar), its insertion in HIV-derived lentiviral vectors clearly augments transduction efficiency (12Follenzi A. Ailles L.E. Bakovic S. Geuna M. Naldini L. Nat. Genet. 2000; 25: 217-222Crossref PubMed Scopus (784) Google Scholar) and nuclear import (13Van Maele B. De Rijck J. De Clercq E. Debyser Z. J. Virol. 2003; 77: 4685-4694Crossref PubMed Scopus (113) Google Scholar). The karyophilic properties of MA and its role in HIV nuclear import (14Bukrinsky M.I. Haggerty S. Dempsey M.P. Sharova N. Adzhubel A. Spitz L. Lewis P. Goldfarb D. Emerman M. Stevenson M. Nature. 1993; 365: 666-669Crossref PubMed Scopus (741) Google Scholar) are unclear (15Reil H. Bukovsky A.A. Gelderblom H.R. Gottlinger H.G. EMBO J. 1998; 17: 2699-2708Crossref PubMed Scopus (213) Google Scholar, 16Fouchier R.A. Meyer B.E. Simon J.H. Fischer U. Malim M.H. EMBO J. 1997; 16: 4531-4539Crossref PubMed Scopus (302) Google Scholar, 17Depienne C. Roques P. Creminon C. Fritsch L. Casseron R. Dormont D. Dargemont C. Benichou S. Exp. Cell Res. 2000; 260: 387-395Crossref PubMed Scopus (91) Google Scholar). Vpr is also not strictly required for HIV replication and DNA integration in non-dividing cells (15Reil H. Bukovsky A.A. Gelderblom H.R. Gottlinger H.G. EMBO J. 1998; 17: 2699-2708Crossref PubMed Scopus (213) Google Scholar, 18Bouyac-Bertoia M. Dvorin J.D. Fouchier R.A. Jenkins Y. Meyer B.E. Wu L.I. Emerman M. Malim M.H. Mol. Cell. 2001; 7: 1025-1035Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 19Zufferey R. Nagy D. Mandel R.J. Naldini L. Trono D. Nat. Biotechnol. 1997; 15: 871-875Crossref PubMed Scopus (1574) Google Scholar). It seems plausible that the DNA flap, MA, and Vpr, albeit redundant, exert additive and/or inter-dependent effects on HIV nuclear import. IN, on the other hand, is an attractive candidate for the role of the PIC import factor. (i) It is essential for the viral replication and spread of infection in primary cells and most T-cell lines (20Nakajima N. Lu R. Engelman A. J. Virol. 2001; 75: 7944-7955Crossref PubMed Scopus (96) Google Scholar); (ii) it is present in the PIC; (iii) its karyophilic properties have been demonstrated by many groups (for references see below). Unfortunately, mutations in IN have pleiotropic effects on viral replication, including alterations in viral particle morphology, defects in reverse transcription and integration (21Engelman A. Adv. Virus Res. 1999; 52: 411-426Crossref PubMed Google Scholar), confounding a detailed genetic analysis of its functions. HIV-1 IN is a 32-kDa protein, initially produced as part of the Gag-Pol precursor polyprotein and released after cleavage by the viral protease during maturation of the virion. IN is responsible for the catalysis of the insertion of the viral DNA into the host cell chromosome (for reviews see Refs. 22Brown P.O. Coffin J.M. Huges S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 161-203Google Scholar, 23Craigie R. J. Biol. Chem. 2001; 276: 23213-23216Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 24Asante-Appiah E. Skalka A.M. Antiviral Res. 1997; 36: 139-156Crossref PubMed Scopus (146) Google Scholar). Like all retroviral INs, HIV-1 IN is composed of three domains. The N-terminal domain harbors an HHCC-type zinc binding domain and has been implicated in the multimerization of the protein (25Lee S.P. Xiao J. Knutson J.R. Lewis M.S. Han M.K. Biochemistry. 1997; 36: 173-180Crossref PubMed Scopus (157) Google Scholar). The core domain contains the catalytic site and possesses structural elements necessary for sequence-specific recognition of the viral long terminal repeat (26Esposito D. Craigie R. EMBO J. 1998; 17: 5832-5843Crossref PubMed Scopus (263) Google Scholar). The arginine/lysine-rich C-terminal domain of IN also contributes to the multimerization of the protein (27Jenkins 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) and is thought to be involved in DNA binding. In addition, binding to DNA has been shown to induce oligomerization of HIV-1 IN in vitro (28Vercammen J. Maertens G. Gerard M. De Clercq E. Debyser Z. Engelborghs Y. J. Biol. Chem. 2002; 277: 38045-38052Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). When expressed in or microinjected into human cells, HIV-1 IN accumulates in the nuclei (17Depienne C. Roques P. Creminon C. Fritsch L. Casseron R. Dormont D. Dargemont C. Benichou S. Exp. Cell Res. 2000; 260: 387-395Crossref PubMed Scopus (91) Google Scholar, 29Pluymers W. Cherepanov P. Schols D. De Clercq E. Debyser Z. Virology. 1999; 258: 327-332Crossref PubMed Scopus (73) Google Scholar, 30Cherepanov P. Pluymers W. Claeys A. Proost P. De Clercq E. Debyser Z. FASEB J. 2000; 14: 1389-1399PubMed Google Scholar, 31Petit C. Schwartz O. Mammano F. J. Virol. 1999; 73: 5079-5088Crossref PubMed Google Scholar, 32Limon A. Devroe E. Lu R. Ghory H.Z. Silver P.A. Engelman A. J. Virol. 2002; 76: 10598-10607Crossref PubMed Scopus (87) Google Scholar, 33Gallay P. Hope T. Chin D. Trono D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9825-9830Crossref PubMed Scopus (420) Google Scholar). During mitosis, IN stably associates with condensed chromosomes (30Cherepanov P. Pluymers W. Claeys A. Proost P. De Clercq E. Debyser Z. FASEB J. 2000; 14: 1389-1399PubMed Google Scholar). Recent studies with digitonin-permeabilized cells have shown that nuclear import of HIV-1 IN can occur in the absence of cytosolic extracts, requires ATP hydrolysis, and is GTPase Ran-independent (34Depienne C. Mousnier A. Leh H. Le Rouzic E. Dormont D. Benichou S. Dargemont C. J. Biol. Chem. 2001; 276: 18102-18107Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Similar observations have been made on the nuclear import of the NLS receptor, importin α (35Miyamoto Y. Hieda M. Harreman M.T. Fukumoto M. Saiwaki T. Hodel A.E. Corbett A.H. Yoneda Y. EMBO J. 2002; 21: 5833-5842Crossref PubMed Scopus (88) Google Scholar). Therefore, the virus either does not rely on the classical importin- and Ran-dependent nuclear import mechanism or is able to take advantage of an alternative pathway. Recently, we have shown that in human cells HIV-1 IN forms a specific nuclear complex with lens epithelium-derived growth factor/transcription co-activator p75 (LEDGF/p75) (36Cherepanov 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 (574) Google Scholar). Recombinant LEDGF/p75 protein effectively promoted HIV-1 IN strand transfer activity in vitro. Of note, LEDGF/p75 was found to be up-regulated in HIV-infected cells (37Schroder A.R. Shinn P. Chen H. Berry C. Ecker J.R. Bushman F. Cell. 2002; 110: 521-529Abstract Full Text Full Text PDF PubMed Scopus (1431) Google Scholar). All these observations suggested that LEDGF/p75 could play a role in retroviral DNA integration. Although its precise cellular function remains elusive, several reports have implicated LEDGF/p75 in gene expression and cellular stress response (38Ge H. Si Y. Roeder R.G. EMBO J. 1998; 17: 6723-6729Crossref PubMed Scopus (254) Google Scholar, 39Fatma N. Singh D.P. Shinohara T. Chylack Jr., L.T. J. Biol. Chem. 2001; 276: 48899-48907Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 40Singh D.P. Fatma N. Kimura A. Chylack Jr., L.T. Shinohara T. Biochem. Biophys. Res. Commun. 2001; 283: 943-955Crossref PubMed Scopus (117) Google Scholar). LEDGF/p75 has been reported to be a DNA-binding protein, with specificity for stress response DNA elements (40Singh D.P. Fatma N. Kimura A. Chylack Jr., L.T. Shinohara T. Biochem. Biophys. Res. Commun. 2001; 283: 943-955Crossref PubMed Scopus (117) Google Scholar). According to the abundant mouse and human LEDGF/p75 mRNA-derived expressed sequence tags in GenBank™, LEDGF/p75 is expressed at all stages of development in a variety of organs and tissues, including skin, bone, thymus, brain, mammary gland, testis, and embryonic and hematopoietic stem cells. A second protein product, p52, can be produced from the same gene as LEDGF/p75 as a result of alternative splicing of the pre-mRNA (38Ge H. Si Y. Roeder R.G. EMBO J. 1998; 17: 6723-6729Crossref PubMed Scopus (254) Google Scholar, 41Singh D.P. Kimura A. Chylack Jr., L.T. Shinohara T. Gene. 2000; 242: 265-273Crossref PubMed Scopus (87) Google Scholar). At least in vitro, p52 was found to be a more general and stronger transcriptional co-activator than LEDGF/p75. In addition, the in vitro interaction of p52 with the ASF/SF2 splicing factor suggests that p52 might play a role in both transcription and splicing (42Ge H. Si Y. Wolffe A.P. Mol. Cell. 1998; 2: 751-759Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In the present work, we demonstrate that HIV-1 IN and LEDGF/p75 are intimate binding partners in human cells and that LEDGF/p75 is crucial for targeting IN to the nucleus and the chromosomes. We also show that both the N-terminal zinc binding and the catalytic core domains of IN are involved in the interaction with LEDGF/p75, whereby the core domain plays the dominant role. EGFP and HcRed1 Fusion Constructs—The full-length IN synthetic gene (INs) (30Cherepanov P. Pluymers W. Claeys A. Proost P. De Clercq E. Debyser Z. FASEB J. 2000; 14: 1389-1399PubMed Google Scholar) or its fragments were PCR-amplified using Pfu DNA polymerase (Stratagene) and an appropriate pair of primers (see below). Each primer contained either a recognition site for XhoI (sense primers) or for HindIII (antisense primers). In-frame stop codons were included in the antisense primers. The primers used were: Cs, 5′-GGGGGCTCGAGCAGACTGCAGAAGCAGATCACC; Cas, 5′-GGGGAAGCTTGGACTTAGTCCTC; Ns, 5′-GGGGGCTCGAGCAGATTCCTGGACGGCATTGAC; Nas, 5′-GGGGAAGCTTACATAGCCTCGCC; Cores, 5′-GGGGGCTCGAGCAGACACGGGCAGGTTGATTGC; Coreas, 5′-GGGGAAGCTTACTCTTTGGTCTGG. The PCR fragments were subcloned between the XhoI and HindIII restriction sites of the pEGFP-C2 vector (Clontech). A series of EGFP fusion constructs were made: pEGFP-INs, coding full-length HIV-1 IN, using the primers Ns and Cas; pEGFP-INs/ΔC, expressing IN with a deletion of the C-terminal domain, using the primers Ns and Coreas; pEGFP-INs/ΔN, expressing IN without the N-terminal zinc binding domain, using the primers Cores and Cas; pEGFP-INs/Nt (using the primers Ns and Nas), pEGFP-INs/Core (using Cores and Coreas), pEGFP-INs/Ct (using Cs and Cas) coding the N-terminal, the central, and the C-terminal IN domain, respectively. The H12N mutant of the synthetic gene was engineered by PCR with the primers H12N (5′-GACGGCATTGACAAGGCTCAGGAGGAGAACGAGAAGTACCACTC) and T3 (5′-AATTAACCCTCACTAAAGGG) using Pwo DNA polymerase (Roche Applied Science) and pINs (30Cherepanov P. Pluymers W. Claeys A. Proost P. De Clercq E. Debyser Z. FASEB J. 2000; 14: 1389-1399PubMed Google Scholar) as the template. The second PCR was performed on the resulting amplicon with the primers Ns and T3; the final PCR product was digested with XhoI and HindIII and subcloned into pEGFP-C2 to obtain pEGFP-INs(H12N). To obtain pHcRed1-INs, for expression of HIV-1 IN fused to the C terminus of the far-red fluorescent protein, HcRed1 (43Fradkov A.F. Verkhusha V.V. Staroverov D.B. Bulina M.E. Yanushevich Y.G. Martynov V.I. Lukyanov S. Lukyanov K.A. Biochem. J. 2002; 368: 17-21Crossref PubMed Scopus (76) Google Scholar), the XcmI/EcoRI restriction fragment of pINs (30Cherepanov P. Pluymers W. Claeys A. Proost P. De Clercq E. Debyser Z. FASEB J. 2000; 14: 1389-1399PubMed Google Scholar) was cloned between the XhoI and EcoRI sites of pHcRed1-C1 (Clontech) after treatment of the XcmI and the XhoI termini of the DNA fragments with T4 DNA polymerase. To generate HcRed1-labeled LEDGF/p75, the BamHI/EcoRI fragment of pCP6H75 (36Cherepanov 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 (574) Google Scholar), spanning the LEDGF/p75 open reading frame, was subcloned between the BglII and EcoRI sites of pHcRed1-C1. The coding sequences of HcRed1 and LEDGF/p75 were placed into frame via BspEI restriction and mung bean nuclease (Invitrogen, Groningen, The Netherlands) digestion followed by re-ligation to obtain the plasmid pHcRed1-p75. To generate an analogous fusion between p52 and HcRed1, the XhoI/EcoRI fragment of pHcRed1-p75 was replaced by the XhoI/EcoRI fragment of pKB-Nat52 (see below), resulting in pHcRed1-p52. The plasmid pEGFP-p75 expressing EGFP-tagged human LEDGF/p75 protein was obtained by inserting the BamHI/EcoRI fragment of pCP6H75 (36Cherepanov 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 (574) Google Scholar) between the BglII and EcoRI sites of pEGFP-C2. Prior to ligation the BamHI and BglII termini of the DNA fragments were filled in, using T4 DNA polymerase. All plasmid constructs used in this work were verified via sequence analysis to confirm absence of mutations. Plasmids for Bacterial Expression of LEDGF/p75, p52, HIV-1 IN, and HIV-1 INH12N—The plasmid pKB-IN6H was used for the expression of the C-terminally tagged form of HIV-1 IN. To obtain pKB-IN6H, the IN gene (derived from the NL4–3 HIV-1 clone) was PCR-amplified from pINSD (44Engelman A. Craigie R. J. Virol. 1992; 66: 6361-6369Crossref PubMed Google Scholar) using the primers 5′-AATACGACTCACTATAGGG (T7 promoter primer) and 5′-GCGCGTCGACATCCTCATCCTGTCTAC (INSalI primer); the resulting PCR fragment was digested with NdeI and SalI and subcloned into the pET-20b(+) vector (Novagen). To create pGM-INH12N-6H, for bacterial expression of the C-terminally His6-tagged INH12N mutant, a DNA fragment containing the HIV-1 IN open reading frame with the mutation was engineered in two consecutive PCR reactions. First, pKB-IN6H was used as the template and a PCR was performed with the forward primer 5′-GATAAGGCCCAAGAAGAAAATGAGAAATATCACAG and the INSalI primer (see above). The resulting amplicon was used as template in the second reaction with the primer 5′-ATATACATATGTTTTTAGATGGAATAGATAAGGCCCAAG and the INSalI primer. The final PCR fragment was digested with NdeI and SalI and cloned into the pET-20b(+) vector. The constructs pCP-Nat75 and pKB-Nat52 were used for bacterial expression of non-tagged LEDGF/p75 and p52 proteins, respectively. A DNA fragment, containing the LEDGF/p75 open reading frame, was amplified using pCP-6H75 (36Cherepanov 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 (574) Google Scholar) as a template and 5′-TGACTCGCGATTTCAAACC and 5′-CCGCGAATTCTAGTTATCTAGTGTAGAATCCTTC as the primers. To obtain the pCP-Nat75 plasmid, this PCR fragment was digested with EcoRI and inserted between the NdeI and EcoRI sites of the pRSETB vector (Invitrogen). Prior to ligation, the NdeI terminus of the vector DNA was filled in using T4 DNA polymerase. To produce pKB-Nat52, a DNA fragment containing the p52 open reading frame was constructed by PCR using the primers 5′-TGACTCGCGATTTCAAACC and 5′-GGCGAATTCTACTGTAGATTACATGTTGTTGGTGCTCAGTTTCCATTTGTTCC. The resulting fragment was digested with EcoRI and cloned between the EcoRI and NdeI sites of pRSETB. Cell Culture and Transfections—HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with GlutaMAX™, 10% fetal calf serum, and 0.02 mg/ml gentamicin (Invitrogen) at 37 °C and 5% CO2 in a humidified atmosphere. Cells were seeded the day before transfection in 8-well LabTek chambered coverglass cuvettes (VWR International, Leuven, Belgium). Transfection of HeLa cells was performed at ∼80% confluency using Lipofectamine 2000 reagent (Invitrogen) with 0.36 μg of plasmid, following the instructions from the manufacturer. Double transfections were accomplished following the same protocol except that transfections were performed with 0.18 μg of each plasmid. For transient LEDGF/p75 knock-down experiments, HeLa cells were transfected with small interfering RNA (siRNA) synthetic duplexes using the Gene Silencer transfection reagent (Gene Therapy Systems) or co-transfected with siRNA plus the pEGFP-INs plasmid using Lipofectamine 2000 according to established protocols (45Elbashir S.M. Harborth J. Weber K. Tuschl T. Methods. 2002; 26: 199-213Crossref PubMed Scopus (1031) Google Scholar). Fetal calf serum was added to the medium at 5 h after transfection. The effect of the RNA interference was studied 48–60 h after transfection. Western Blotting and Indirect Immunofluorescence—For Western blot detection, protein extracts separated in 11% SDS-PAGE gels were electroblotted onto polyvinylidene difluoride membranes (Bio-Rad, Nazareth, Belgium). The polyclonal anti-GFP antibody was purchased from Invitrogen; the mouse anti-LEDGF p75/p52 antibody was from BD Biosciences (Erembodegem, Belgium). The polyclonal rabbit anti-HIV-1 IN antibody has been described previously (30Cherepanov P. Pluymers W. Claeys A. Proost P. De Clercq E. Debyser Z. FASEB J. 2000; 14: 1389-1399PubMed Google Scholar). Secondary horseradish peroxidase- or alkaline phosphatase-conjugated goat anti-mouse and goat anti-rabbit antibodies were from Dako Diagnostics (Leuven, Belgium). Detection was carried out using ECL+ chemiluminescent horseradish peroxidase substrate (Amersham Biosciences) or with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium chromogenic substrate for alkaline phosphatase. The broad range prestained protein marker mix (New England Biolabs, Hertfordshire, United Kingdom) was used for estimation of the molecular weights. Indirect immunofluorescent detection of endogenous LEDGF/p75 was performed as previously described (36Cherepanov 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 (574) Google Scholar). Cells grown in LabTek II glass chamber slides (VWR International) were fixed by incubation with 4% formaldehyde in PBS for 10 min, washed with PBS, and permeabilized/fixed with ice-cold methanol. The cells were then blocked in 10% fetal calf serum, 20 mm ammonium chloride, and PBS for 30 min and incubated with monoclonal anti-LEDGF p75/p52 antibodies (used at a dilution of 1:300), followed by Alexa-488- or Alexa-568-conjugated goat anti-mouse antibody (Molecular Probes, Leiden, The Netherlands). The nuclear DNA was stained with 5 μm ToPro3 iodide (Molecular Probes). Laser Scanning Microscopy and Image Analysis—Confocal microscopy was performed using an LSM 510 unit (Zeiss, Zaventem, Belgium). SYTO 17 (Molecular Probes) was used to stain DNA of live cells. Prior to image acquisition, cells were washed with serum-free OptiMEM (Invitrogen). All two- and three-color images with a resolution of 1024 × 1024 pixels were acquired in the multi-track mode. EGFP was excited at 488 nm (AI laser), HcRed1 at 543 nm (HeNe laser), and SYTO 17 at 633 nm (HeNe laser). After the main dichroic beam splitter (HFT 488/543 for EGFP and HcRed1, HFT 488/543/633 for EGFP and SYTO 17) the fluorescence signal was divided by a secondary dichroic beam splitter (NFT 635 VIS or NFT 543) and detected in the separate channels using the appropriate filters: BP 505–530 (for EGFP), LP560 (for HcRed1), and LP650 (for SYTO 17). In this set-up, no cross-talk between the green and red channels was observed. The colocalization of fluorescently-tagged proteins in the nucleus was quantified using the Image-Pro Plus version 4.5 software (Media Cybernetics, Carlsbad, CA) and expressed in terms of the correlation coefficient (rp) (same as Pearson's r correlation) (46Gonzalez R.C. Woods R.E. Digital Image Processing. 2nd Ed. Prentice Hall, Upper Saddle River, NJ2002: 693-753Google Scholar). rp=∑i((S1i-〈S1〉)·(S2i-〈S2〉))∑i(S1i-〈S1〉)2·∑i(S2i-〈S2〉)2(Eq. 1) S1 and S2 represent the signal intensities of pixels in the first and second channel, respectively; 〈S1〉 and 〈S2〉 are the average intensity of the first channel and second channel, respectively. The correlation coefficient is a value between –1 and +1, with –1 corresponding to negative correlation between images and +1 corresponding to a total overlap of the images from the two channels. It reflects similarity of image patterns and does not depend on intensities of the images. Recombinant Proteins—Non-tagged LEDGF/p75 and p52 proteins were produced from the plasmids pCP-Nat75 and pKB-Nat52, respectively, in the Endo I-free PC1 Escherichia coli host strain (E. coli B, BL21(DE3), ΔendA::TcR, pLysS) (47Cherepanov 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). Expression was induced in LB medium at 29 °C by addition of 0.5 mm isopropyl-1-thio-β-d-galactopyranoside. Cells harvested 3 h after induction were disrupted using a French press in 450 mm NaCl, 30 mm Tris, pH 7.0. The supernatant obtained by centrifugation of the lysate was passed through a 1-ml HiTrap heparin column (Amersham Biosciences, Uppsala, Sweden) to capture LEDGF/p75 or p52, and the protein was eluted by a linear gradient of NaCl concentration in 30 mm Tris, pH 7.0. The fractions containing LEDGF/p75 or p52 were pooled and further purified by cation exchange chromatography on a 1-ml HiTrap SP Sepharose column (Amersham Biosciences). To produce C-terminally His6-tagged wild type HIV-1 IN, PC1 E. coli cells harboring pKB-IN6H were grown in LB medium to an optical density of 0.8 and induced by addition of 0.5 mm isopropyl-1-thio-β-d-galactopyranoside, at 29 °C for 3 h. The protein was purified essentially as described for N-terminally tagged HIV-1 IN (48Cherepanov P. Este J.A. Rando R.F. Ojwang J.O. Reekmans G. Steinfeld R. David G. De Clercq E. Debyser Z. Mol. Pharmacol. 1997; 52: 771-780Crossref PubMed Scopus (95) Google Scholar). In brief, cells were lysed using a French press in 1 m NaCl, 7.5 mm CHAPS, 30 mm Tris, pH 7.4, and the soluble His6-tagged IN protein was enriched by batch adsorption to Ni-NTA-agarose (Qiagen, Hilden, Germany). Protein eluted with 200 mm imidazole, 1 m NaCl, 7.5 mm CHAPS, 30 mm Tris, pH 7.4 was further purified on a 1-ml HiTrap heparin column (Amersham Biosciences). The His6-tagged H12N mutant was induced in PC1 cells from pGM-INH12N-6H and purified in a similar way. Purified recombinant LEDGF/p75, p52, IN, and INH12N proteins were concentrated by ultrafiltration using Centricon 10 (Millipore, Brussels, Belgium), supplemented with 5 mm dithiothreitol plus 10% glycerol, and kept frozen at –80 °C. His6 Tag Integrase Pull-down Assay—Binding of IN to LEDGF/p75 or p52 was assayed in 25 mm Tris-HCl, pH 7.4, 0.1% Nonidet P-40, 20 mm imidazole containing 100 or 400 mm NaCl, in the presence or absence of 1 mm MgCl2 (binding buffer). 1 μg of recombinant His6-tagged HIV-1 IN or His6-INH12N was incubated with 1–3 μg of LEDGF/p75 or p52 in 200 μl of binding buffer supplemented with 2 μg of bovine serum albumin (BSA). Follo" @default.
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- W2088335428 title "LEDGF/p75 Is Essential for Nuclear and Chromosomal Targeting of HIV-1 Integrase in Human Cells" @default.
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