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• W2091216865 abstract "The human immunodeficiency virus (HIV-1) nucleocapsid protein NCp7 containing two CX 2CX 4HX 4C-type zinc fingers was proposed to be involved in reverse transcriptase (RT)-catalyzed proviral DNA synthesis through promotion of tRNA3Lys annealing to the RNA primer binding site, improvement of DNA strand transfers, and enhancement of RT processivity. The NCp7 structural characteristics are crucial because mutations altering the finger domain conformation led to noninfectious viruses characterized by defects in provirus integration. These findings prompted us to study a putative RT/NCp7 protein-protein interaction. Binding assays using far Western analysis or RT immobilized on beads clearly showed the formation of a complex between NCp7 and RT. The affinity of NCp7 for p66/p51RT was 0.60 μm with a 1:1 stoechiometry. This interaction was confirmed by chemical cross-linking and co-immunoprecipitation of the two proteins in a viral environment. Competition experiments using different NCp7 mutants showed that alteration of the finger structure disrupted RT recognition, giving insights into the loss of infectivity of corresponding HIV-1 mutants. Together with structural data on RT, these results suggest that the role of NCp7 could be to enhance RT processivity through stabilization of a p51-induced active form of the p66 subunit and open the way for designing new antiviral agents. The human immunodeficiency virus (HIV-1) nucleocapsid protein NCp7 containing two CX 2CX 4HX 4C-type zinc fingers was proposed to be involved in reverse transcriptase (RT)-catalyzed proviral DNA synthesis through promotion of tRNA3Lys annealing to the RNA primer binding site, improvement of DNA strand transfers, and enhancement of RT processivity. The NCp7 structural characteristics are crucial because mutations altering the finger domain conformation led to noninfectious viruses characterized by defects in provirus integration. These findings prompted us to study a putative RT/NCp7 protein-protein interaction. Binding assays using far Western analysis or RT immobilized on beads clearly showed the formation of a complex between NCp7 and RT. The affinity of NCp7 for p66/p51RT was 0.60 μm with a 1:1 stoechiometry. This interaction was confirmed by chemical cross-linking and co-immunoprecipitation of the two proteins in a viral environment. Competition experiments using different NCp7 mutants showed that alteration of the finger structure disrupted RT recognition, giving insights into the loss of infectivity of corresponding HIV-1 mutants. Together with structural data on RT, these results suggest that the role of NCp7 could be to enhance RT processivity through stabilization of a p51-induced active form of the p66 subunit and open the way for designing new antiviral agents. The HIV-1 1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; DTT, dithiothreitol; DTSSP, 3,3′-dithiobis[sulfosuccinimidyl propionate].1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; DTT, dithiothreitol; DTSSP, 3,3′-dithiobis[sulfosuccinimidyl propionate]. nucleocapsid protein NCp7 is a highly basic protein containing two zinc fingers of the CX 2CX 4HX 4C type (Fig. 1) found in tight association with the dimeric RNA genome in the retrovirus core (1Coffin J.M. Field B.N. Knippe D.M. Field B.N. Knippe D.M. 2nd Ed. Retroviridae and Their Replication: Virology. 51. Raven Press, Ltd, New York1990: 1437-1500Google Scholar). In vivo, NCp7 is required for the protection of the genome against cellular nucleases and is involved in genomic RNA packaging and morphogenesis of virus particles (review in Ref. 2Darlix J.L. Lapadat-Tapolsky M. de Rocquigny H. Roques B.P. J. Mol. Biol. 1995; 254: 523-537Crossref PubMed Scopus (381) Google Scholar). Most of these functions are related to its well demonstrated high affinity for single-stranded nucleic acids (3Khan R. Giedroc D.P. J. Biol. Chem. 1992; 267: 6689-6695Abstract Full Text PDF PubMed Google Scholar). NMR studies have demonstrated that the folded CCHC boxes of NCp7 are in spatial proximity, whereas the N- and C-terminal sequences remain flexible (4Morellet N. Jullian N. de Rocquigny H. Maigret B. Darlix J.L. Roques B.P. EMBO J. 1992; 11: 3059-3065Crossref PubMed Scopus (217) Google Scholar,5Lee B.M. de Guzman R.N. Turner B.G. Tjandra N. Summers M.F. J. Mol. Biol. 1998; 279: 633-649Crossref PubMed Scopus (134) Google Scholar). Mutations inducing modifications in the general conformation of the protein, such as the replacement of His23 by Cys, Pro31 by Leu, or Trp37 by a nonaromatic residue led to more or less important defects in RNA packaging and virus core morphology (6Déméné H. Dong C.Z. Ottmann M. Rouyez M.C. Jullian N. Morellet N. Mély Y. Darlix J.L. Fournié-Zaluski M.C. Saragosti S. Roques B.P. Biochemistry. 1994; 33: 11707-11716Crossref PubMed Scopus (101) Google Scholar, 7Morellet N. de Rocquigny H. Mély Y. Jullian N. Déméné H. Ottmann M. Gérard D. Darlix J.L. Fournié-Zaluski M.C. Roques B.P. J. Mol. Biol. 1994; 235: 287-301Crossref PubMed Scopus (136) Google Scholar, 8Aldovini A. Young R.A. J. Virol. 1990; 64: 1920-1926Crossref PubMed Google Scholar, 9Dorfmann T. Luban I. Goff S.P. Haseltine W.A. Göttlinger H.G. J. Virol. 1993; 67: 6159-6169Crossref PubMed Google Scholar, 10Gorelick R.J. Nigida Jr., S.M. Bess Jr., J.W. Arthur L.O. Henderson L.E. Rein A. J. Virol. 1990; 64: 3207-3211Crossref PubMed Google Scholar, 11Ottmann M. Gabus C. Darlix J.L. J. Virol. 1995; 69: 1778-1784Crossref PubMed Google Scholar), which seem hardly reconcilable with the complete loss of infectivity of the mutated viruses. One possible explanation could be that changes in NCp7 structure hinders one essential NCp7-dependent step of virus life cycle such as reverse transcription and provirus synthesis (12Yu Q. Darlix J.L. J. Virol. 1996; 70: 5791-5798Crossref PubMed Google Scholar, 13Tanchou V. Décimo D. Péchoux C. Lener D. Rogemond V. Berthoux L. Ottmann M. Darlix J.L. J. Virol. 1998; 72: 4442-4447Crossref PubMed Google Scholar, 14Cameron C.E. Ghosh M. Le Grice S.F.J. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6700-6705Crossref PubMed Scopus (77) Google Scholar). This process is catalyzed by the p66/p51 heterodimeric virion-associated reverse transcriptase (RT). This enzyme exhibits RNA- and DNA-dependent DNA polymerase activities and an RNase H activity, which are achieved by the p66 polypeptide chain (review in Ref. 15Katz R.A. Skalka A.M. Annu. Rev. Biochem. 1994; 63: 133-173Crossref PubMed Scopus (534) Google Scholar). In vitro, HIV-RT shows an unusual low processivity for a replicative enzyme, suggesting that additional factors are required for efficient viral DNA synthesis in vivo. In vitro, NCp7 has been shown to activate the annealing of the replication primer tRNA3Lys at the initiation site of reverse transcription (16Barat C. Lullien V. Schatz O. Keith G. Nugeyre M.T. Grüninger-Leitch F. Barré-Sinoussi F. Le Grice S.F. Darlix J.L. EMBO J. 1989; 8: 3279-3285Crossref PubMed Scopus (261) Google Scholar, 17Remy E. de Rocquigny H. Petitjean P. Muriaux D. Theilleux V. Paoletti J. Roques B.P. J. Biol. Chem. 1998; 273: 4819-4822Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and the 5′-3′ viral DNA strand transfer, leading to provirus formation (18Darlix J.L. Vincent A. Gabus C. de Rocquigny H. Roques B.P. C. R. Acad. Sci. (Paris). 1993; 316: 763-771PubMed Google Scholar, 19Rodriguez-Rodriguez L. Tsuchihashi Z. Fuentes G.M. Bambara R.A. Fay P.J. J. Biol. Chem. 1995; 270: 15005-15011Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 20DeStefano J.J. J. Biol. Chem. 1996; 271: 16350-16356Abstract Full Text Full Text PDF PubMed Google Scholar, 21Guo J. Henderson L.E. Bess J. Kane B. Levin J.G. J. Virol. 1997; 71: 5178-5188Crossref PubMed Google Scholar). NCp7 was shown to reduce nonspecific reverse transcription (18Darlix J.L. Vincent A. Gabus C. de Rocquigny H. Roques B.P. C. R. Acad. Sci. (Paris). 1993; 316: 763-771PubMed Google Scholar, 21Guo J. Henderson L.E. Bess J. Kane B. Levin J.G. J. Virol. 1997; 71: 5178-5188Crossref PubMed Google Scholar, 22Li X. Quan Y. Arts E.J. Li Z. Preston B.D. de Rocquigny H. Roques B.P. Darlix J.L. Kleiman L. Parniack M.A. Wainberg M.A. J. Virol. 1996; 70: 4996-5004Crossref PubMed Google Scholar) and to enhance the efficiency and processivity of RT (19Rodriguez-Rodriguez L. Tsuchihashi Z. Fuentes G.M. Bambara R.A. Fay P.J. J. Biol. Chem. 1995; 270: 15005-15011Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 23Peliska J.A. Balasubramanian S. Giedroc D.P. Benkovic J.A. Biochemistry. 1994; 33: 13817-13823Crossref PubMed Scopus (161) Google Scholar, 24Wu W. Henderson L.E. Copeland T.D. Gorelick R.J. Bosche W.J. Rein A. Levin J.G. J. Virol. 1996; 70: 7132-7142Crossref PubMed Google Scholar, 25Ji X. Klarmann G.J. Preston B.D. Biochemistry. 1996; 35: 132-143Crossref PubMed Scopus (116) Google Scholar, 26Tanchou V. Gabus C. Rogemond V. Darlix J.L. J. Mol. Biol. 1995; 252: 563-571Crossref PubMed Scopus (112) Google Scholar), suggesting that it could activate reverse transcription through direct interaction with the enzyme (18Darlix J.L. Vincent A. Gabus C. de Rocquigny H. Roques B.P. C. R. Acad. Sci. (Paris). 1993; 316: 763-771PubMed Google Scholar, 20DeStefano J.J. J. Biol. Chem. 1996; 271: 16350-16356Abstract Full Text Full Text PDF PubMed Google Scholar, 22Li X. Quan Y. Arts E.J. Li Z. Preston B.D. de Rocquigny H. Roques B.P. Darlix J.L. Kleiman L. Parniack M.A. Wainberg M.A. J. Virol. 1996; 70: 4996-5004Crossref PubMed Google Scholar, 23Peliska J.A. Balasubramanian S. Giedroc D.P. Benkovic J.A. Biochemistry. 1994; 33: 13817-13823Crossref PubMed Scopus (161) Google Scholar, 24Wu W. Henderson L.E. Copeland T.D. Gorelick R.J. Bosche W.J. Rein A. Levin J.G. J. Virol. 1996; 70: 7132-7142Crossref PubMed Google Scholar, 26Tanchou V. Gabus C. Rogemond V. Darlix J.L. J. Mol. Biol. 1995; 252: 563-571Crossref PubMed Scopus (112) Google Scholar, 27Drummond J.E. Mounts P. Gorelick R.J. Casas-Finet J.R. Bosche W.J. Henderson L.E. Waters D.J. Arthur L.O. AIDS Res. Hum. Retroviruses. 1997; 13: 533-543Crossref PubMed Scopus (35) Google Scholar). Accordingly, NCp7 was shown to be capable of re-establishing strand transfer efficiency and RNase H activity of a defective RT mutant (14Cameron C.E. Ghosh M. Le Grice S.F.J. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6700-6705Crossref PubMed Scopus (77) Google Scholar). In this study, RT and NCp7 have been shown for the first time to form a 1:1 complex characterized by an affinity of 6.0 × 10−7m. The structure-activity study has emphasized the critical role of the zinc finger domain in the complexation, giving insights into the loss of virus infectivity of mutants with structurally altered NCp7. Co-immunoprecipitation of NCp7 and RT in a viral environment and cross-linking experiments suggest that NCp7 could enhance RT processivity through stabilization by its zinc finger domain of the p51-induced active form of p66 RNase H domain (28Hostomsky Z. Hostomska Z. Hudson G.O. Moomaw E.W. Nodes B.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1148-1152Crossref PubMed Scopus (94) Google Scholar). Moreover, the N-terminal part of NCp7 could compensate for the absence in RNase H domain of HIV-1 p66 RT of the sequence present in the Escherichia coli RT (29Davies J.F. Hostomska Z. Hostomsky Z. Jordan S.R. Matthews D.A. Science. 1991; 252: 88-95Crossref PubMed Scopus (525) Google Scholar). The critical role of the complex during viral DNA synthesis suggests that inhibition of this protein-protein interaction could be an interesting way for designing new antiviral agents. NCp7, (12–53)NCp7, W16F37(12–53)NCp7, L37(12–53)NCp7, C23(12–53)NCp7, (a-D)NCp7, which corresponds to a peptide in which the zinc finger domains have been replaced by two Gly-Gly linkers (see Fig. 1), and Vpr were synthesized on a 433 automated peptide synthesizer (Applied Biosystem) using the procedure already described (30de Rocquigny H. Gabus C. Vincent A. Fournié-Zaluski M.C. Roques B.P. Darlix J.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6472-6476Crossref PubMed Scopus (279) Google Scholar, 31de Rocquigny H. Petitjean P. Tanchou V. Décimo D. Drouot L. Delaunay T. Darlix J.L. Roques B.P. J. Biol. Chem. 1997; 272: 30753-30759Crossref PubMed Scopus (72) Google Scholar). Anti-NCp7 mouse monoclonal antibodies (mAb) were either HH3 (32Tanchou V. Delaunay T. de Rocquigny H. Bodeus M. Darlix J.L. Roques B.P. Benarous R. AIDS Res. Hum. Retroviruses. 1994; 10: 983-993Crossref PubMed Scopus (18) Google Scholar) or 2B10, 2H. de Rocquigny, A. Caneparo, C. Z. Dong, P. Petitjean, T. Delaunay, and B. P. Roques, submitted for publication. which are directed against the C-terminal part (52–67) of NCp7 and the first zinc finger of NCp7, respectively. Rabbit polyclonal antibody against RT was a generous gift from S. Litvak. Monoclonal antibody against Vpr was obtained from the synthetic peptide (31de Rocquigny H. Petitjean P. Tanchou V. Décimo D. Drouot L. Delaunay T. Darlix J.L. Roques B.P. J. Biol. Chem. 1997; 272: 30753-30759Crossref PubMed Scopus (72) Google Scholar). HIV-1 (NL43 strain) was produced by transfecting plasmid pNL43 into 293T cells using the calcium phosphate precipitation standard procedure. This virus was pseudotyped with the vesicular stomatotitis virus G glycoprotein by co-transfection of vesicular stomatotitis virus G glycoprotein-encoding plasmid. Enzyme-linked immunosorbent assay (kit from E. I. du Pont de Nemours & Co.) was carried out to quantify p24 content of the viral stock. The infectivity of the virus was determined on HeLa cells as described (33Schwartz O. Dautry-Varsat A. Goud B. Marechal V. Subtil A. Heard J.M. Danos O. J. Virol. 1995; 69: 528-533Crossref PubMed Google Scholar). After ultracentrifugation (50,000 ×g), the virus was resuspended in 200 μl of phosphate buffer, pH 7.5, containing 150 mm NaCl and 0.5% Triton. The concentrations of viral proteins in the sample were 30 μg of p24/ml, 10 μg of NCp7/ml, and 3.5 μg of RT/ml. 2 μg of purified HIV-1 p66/p51 RT (Worthington, Freehold, New Jersey) were immobilized onto a Hybond-C super nitrocellulose membrane (Amersham Pharmacia Biotech) in 100 μl of TBS (25 mm Tris, 100 mm NaCl, 0.1 mm DTT, 3 mm KCl, pH 7.4) for 3 h at room temperature. The membrane was treated with SuperBlock blocking buffer (Pierce) for 3 h at room temperature to reduce nonspecific interactions and then incubated overnight at 4 °C either with NCp7, (a-D)NCp7, (12–53)NCp7, or Vpr (2.1 μm) or without proteins as a control in 4 ml of 5% dry milk in TBS buffer containing 0.1% Tween 20. After three washes in TBS-Tween and a 45-min incubation in superblocker, blots were revealed by incubation, first, with 1/15,000 anti-NCp7 mAb (HH3 for NCp7 and (a-D)NCp7, 2B10 for (12–53)NCp7) or 1/1000 anti-Vpr mAb for 3 h in 5% dry milk TBS-Tween followed by treatment with peroxidase-conjugated anti-mouse antibody for 1 h. These antibodies were not able to cross-react with RT. Complexes were revealed by the ECL method (Amersham Pharmacia Biotech) with peroxidase substrate incubation. Alternatively, 2 μg of HIV-RT were loaded on a 15% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred onto nitrocellulose membrane. The blot was incubated with 2.1 μm NCp7 or without NCp7 as control following the procedure described above using HH3 mAb against NCp7. HIV-1 RT was covalently linked to CNBr-activated Sepharose 4B beads (Amersham Pharmacia Biotech) using the standard procedure described by the supplier. Typically, 100 mg of CNBr-activated Sepharose beads were swollen and washed in 20 ml of HCl (1 mm), then equilibrated in coupling buffer (0.1m NaHCO3, 500 mm NaCl, pH 8.3), thus providing 350 μl of gel, which was then diluted in 350 μl of coupling buffer. The batch was divided in two equal parts. 40 μg of HIV-1 RT in 200 μl of coupling buffer were added to the first batch, and only coupling buffer was added to the second one. After a night at 4 °C, excess ligand was washed away, and remaining unbound sites were inactivated in 1 m 1,3-diaminopropane, pH 8, during 2 h at room temperature. The two bead batches were washed with 3 5-min cycles of alternating pH using 0.1 m acetate buffer (500 mm NaCl, pH 4) and 0.1 m Tris-HCl buffer (500 mm NaCl, pH 8). The efficiency of RT immobilization was checked by detecting the quantity of unbound proteins remaining in the supernatant using Western blot analysis and calculated to be superior to 98% (data not shown), suggesting that 0.23 μg (2 × 10−12 mol) of RT has been captured by μl of beads. Then, 20 μl of these beads corresponding to about 4.6 μg of protein (3.9 × 10−11 mol) were equilibrated in TBS, 0.1% Tween 20, 0.1% Nonidet P-40 and incubated with increasing concentrations of NCp7 from 0.1 up to 4.2 μm in 100 μl of the same buffer for 5 h at room temperature. After centrifugation (6500 rpm), the beads were washed twice with the previous cold buffer at 4 °C. NCp7 bound to immobilized RT or nonspecifically to the beads was recovered by heating at 80 °C for 5 min in 30 μl of Laemmli buffer (50 mm Tris, 10% glycerol, 2% SDS, 0.05% bromphenol blue, 200 mm DTT). Collected samples were loaded on a 20% SDS-PAGE, transferred onto nitrocellulose, and analyzed by Western blot using HH3 mAb in order to reveal NCp7. The effect of NaCl concentration on NCp7/RT complex formation was measured using the same procedure, except that the beads were incubated with a constant concentration of NCp7 (0.9 μm) in TBS-Tween-Nonidet P-40 buffer containing 100 to 500 mm NaCl. When (12–53)NCp7 or its derivatives were used as competitors, two concentrations of these peptides (4 and 20 μm) were preincubated for 90 min with immobilized RT before the addition of 0.4 μm NCp7. In this case, the monoclonal antibody used (HH3) was selective for NCp7 and was unable to cross-react with (12–53)NCp7 or its derivatives. The affinity and stoichiometry of the NCp7/RT complex were determined by quantification of NCp7 bound to immobilized RT or nonspecifically bound to the beads using a standard curve of pure NCp7 and a Bio-profile imager (Vilber-Lourmat). Nonspecific binding was subtracted from the total binding, thus enabling us to calculate the concentrations of bound and free NCp7. The binding parameters were calculated using the Scatchard equation from Enzfit software. Results are means of three independent experiments performed in duplicate. Surface plasmon resonance experiments were carried out on an Amersham Pharmacia Biotech BIAcore 2000 apparatus. CM5 sensorchips,N-hydroxysuccinimide,N-ethyl-N′-(dimethylaminopropyl)carbodiimide, and surfactant p20 were supplied by Amersham Pharmacia Biotech. 1,3-Diaminopropane was from Aldrich. CM5 sensorchip was stabilized in HBS running buffer (10 mm Hepes, 150 mm NaCl, 0.005% p20, pH 7.4) and activated using theN-ethyl-N′-(1,3-diethylamide-propyl)carbodiimide/N-hydroxysuccinimide procedure described by Amersham Pharmacia Biotech. Then, about 6000 response units of RT were reproducibly bound following a 20-μl pulse (5 μl/min) with a solution of RT protein (0.1 μg/μl) diluted in sodium acetate buffer, pH 4.5. To prevent possible electrostatic interactions with (12–53)NCp7 and its derivatives, remaining activated carboxyl groups were blocked by the addition of 1,3-diaminopropane (1 mm, 30 μl). The bulk refractive index due to injected proteins was corrected using a nonderivatized channel as control. Binding experiments were performed at 25 °C with a 20 μl/min flow rate in HBS running buffer (50 mm NaCl). 50 μl of (12–53)NCp7, W16F37(12–53)NCp7, L37(12–53)NCp7, and C23(12–53)NCp7 solutions (1.6 μm) were injected three times on immobilized RT and on the control channel. Flow cells were regenerated by a 5-μl pulse of 0.05% SDS in HBS running buffer followed by 2.5 m NaCl solution. RT and NCp7 were cross-linked with a homobifunctionalN-hydroxysuccinimide ester-conjugated reagent DTSSP (Pierce), leading to covalent bonds between amino groups of both proteins. After incubation of 1 μg of RT and 4.6 μg of NCp7 (40 equivalents) for 1 h at room temperature in 10 μl of phosphate-buffered saline (3 mm KCl, 1.5 mmKH2PO4, 140 mm NaCl, 8 mm Na2HPO4, pH 7.5), DTSSP was added (2 or 5 mm) for 30 min. The cross-linking reaction was stopped by incubating the mixture for 30 min in 50 mmTris, 6 mm glycine buffer. Proteins boiled in Laemmli buffer without DTT were resolved by electrophoresis on a 12% SDS-PAGE, transferred onto nitrocellulose membrane, and analyzed by two successive Western blots using, first, rabbit polyclonal antibody against RT (a generous gift from S. Litvak) and then, after dehybridation in 0.1 m CH3COOH, a mouse mAb against NCp7 (HH3) as described above. 50 μl of crude extract obtained from 293T transfected cells were rocked with 50 μl of ascite fluid containing mAb against NCp7 (HH3 or 2B10) overnight at 4 °C. Then, 25 μl of protein G-Sepharose beads (Amersham Pharmacia Biotech) equilibrated in 80 mm Tris, 80 mm NaCl, pH 8.0, 1% Nonidet P-40 were added, and the suspension was incubated for 4 h at 4 °C. Beads were collected by centrifugation (10,000 ×g for 5 s) and washed at 4 °C twice with the previous buffer and once with a low salt buffer (80 mmTris, pH 8.0, 0.1% Nonidet P-40, 0.05% sodium deoxycholate). Beads were boiled in Laemmli buffer. Proteins were resolved by electrophoresis on a 15% SDS-PAGE and analyzed by two successive Western blots in order to reveal RT and NCp7 using the previously mentioned procedure. Interactions between NCp7 and heterodimeric p66/p51RT or monomeric p66RT and p51RT forms alone were investigated in vitro by far Western blot analysis using RT samples immobilized on nitrocellulose membrane under nondenaturating (Fig. 2 A) or denaturating conditions (Fig. 2 B). The formation of a complex was evidenced using a mAb directed against NCp7 or its derivatives, which is unable to cross-react with native RT. As depicted in Fig. 2 A, NCp7 (1st lane) and its central zinc-fingered domain, (12–53)NCp7 (3rd lane), interact with the heterodimeric RT, whereas (a-D)NCp7 (2ndlane), in which both zinc fingers have been replaced by two Gly-Gly linkers (Fig. 1), was found unable to bind RT. In contrast, even after a long exposure time, no signal could be detected when Vpr, another basic HIV-1 protein (31de Rocquigny H. Petitjean P. Tanchou V. Décimo D. Drouot L. Delaunay T. Darlix J.L. Roques B.P. J. Biol. Chem. 1997; 272: 30753-30759Crossref PubMed Scopus (72) Google Scholar), was used instead of NCp7 (Fig.2 A, 4th lane). Likewise, p6, the second maturation product of p15, was found unable to recognize RT (data not shown). Fig. 2 B shows that NCp7 recognized both subunits of RT, independently. This interaction is not critically dependent on salt concentration, because only a slight difference was observed when binding experiments were performed using buffers containing 100 up to 500 mm NaCl (Fig.2 C). The affinity of NCp7 for RT was measured by means of an affinity test based on the immobilization of p66/p51RT onto Sepharose beads through the formation of covalent bonds between amino groups of RT and CNBr-preactivated sites on the resin. The beads were incubated with increasing concentrations of NCp7 from 0.1 up to 4.2 μmor without NCp7 as control. After washes, the bound NCp7 molecules were released from the beads by denaturation and analyzed by successive 20% SDS-PAGE, nitrocellulose membrane transfer and Western blot analysis with NCp7 mAb (32Tanchou V. Delaunay T. de Rocquigny H. Bodeus M. Darlix J.L. Roques B.P. Benarous R. AIDS Res. Hum. Retroviruses. 1994; 10: 983-993Crossref PubMed Scopus (18) Google Scholar). Fig. 3 A, which represents the total binding of NCp7 on immobilized RT, confirmed the formation of a dose-dependent RT/NCp7 complex, previously characterized by far Western analysis. The nonspecific binding of NCp7 (Fig. 2 C, 1st lane) was significantly reduced by adding 1,3-diaminopropane to the RT substituted resin, which blocked the remaining CNBr-activated groups and provided positive charges at the surface of the beads, resulting in electrostatic repulsion of the positively charged NCp7. The specific saturation curve and the binding parameters were determined from Scatchard representation. The calculated apparent affinityK D was 0.60 μm (± 0.07) for a 1:1 stoichiometry (n = 0.86 ± 0.04), corresponding to about 1 NCp7 molecule for 1 molecule of the heterodimer p66/p51RT (Fig.3, B and C). Since it appears from the experiments with (a-D)NCp7 (Fig. 2 A) that the N- and C-terminal parts of NCp7 are not critical for RT recognition, the contribution of the zinc finger domains was investigated by measuring the direct binding of (12–53)NCp7 and its various mutants, W16F37(12–53)NCp7, L37(12–53)NCp7, and C23(12–53)NCp7, to RT using surface plasmon resonance experiments. One concentration (1.6 μm) of each peptide was injected to 6000 response units of RT immobilized on the sensorchip in HBS buffer (10 mmHepes, 50 mm NaCl, 0.005% p20, pH 7.4). As shown on Fig.4 A, (12–53)NCp7 was able to recognize RT. The inversion of aromatic residues in W16F37(12–53)NCp7 induced a 50% decrease of RT binding as compared with wild-type (12–53)NCp7. Moreover, the replacement of Trp37 by Leu or His23 by Cys dramatically disturbed the RT recognition, because in both cases, only 20% of wild-type binding was retained. These results were confirmed by competition experiments using immobilized RT on Sepharose beads. For this purpose, the beads were preincubated with two different concentrations of (12–53)NCp7 or various mutants. Wild-type NCp7 was then added at 0.4 μm, and the collected samples were analyzed by Western blot as above in order to quantify the amount of NCp7 bound to RT. The monoclonal antibody used (HH3) interacts selectively with NCp7 and is unable to cross-react with the competing NCp7 derivatives. The addition of 10 equivalents of (12–53)NCp7 induced a complete disappearance of the NCp7 band (Fig.4 B, lanes 3–4), supporting the importance of the finger domain in RT recognition. Moreover, the structure of the zinc finger domain appears important for the interaction with RT because W16F37(12–53)NCp7 appeared to be able to decrease NCp7 binding only at 50 equivalents (Fig. 4 B,lanes 9–10), whereas L37(12–53)NCp7 (lanes 5–6) and C23(12–53)NCp7 (lanes 7–8) were unable to impede RT/NC recognition. The interaction between RT and NCp7 was confirmed by cross-linking experiments using 2 and 5 mm DTSSP, which cross-links compounds in close spatial proximity by formation of covalent amide bonds with amino groups of interacting proteins (Fig. 5,A and B). Therefore, NCp7 and RT were preincubated to allow the complex to be preformed. Then DTSSP was added. After quenching the reaction with Tris-glycine buffer, samples were analyzed by two successive Western blots using antibodies directed either against RT (Fig. 5 A) or against NCp7 (Fig.5 B). As expected, this resulted in the formation of various species with apparent molecular masses of 59, 74, 117, and 134 kDa that reacted with antibodies directed against RT (Fig. 5 A). The band at 117 kDa corresponds to the coupling of the p51 and p66 subunits. When anti-NCp7 mAb was used (Fig. 5 B), NCp7 could be detected on the previously mentioned adducts migrating at the 59-, 74-, and 134-kDa positions. The first and second compounds correspond to the covalent binding of NCp7 to the p51 and p66 subunits of RT, respectively, and the third one to the binding of two molecules of NCp7 to p66/p51 RT. Interestingly, the p66/NCp7 cross-linked compound is the most abundant adduct. Weak bands at 16, 24, and 32 kDa, corresponding to dimers, trimers, and tetramers of NCp7, were observed. The existence of a RT/NCp7 complex was confirmed by co-immunoprecipitation using a crude extract from 293T cells transfected by pNL43 HIV-1 plasmid. The viral preparation was treated with HH3 or 2B10 mouse monoclonal antibody. Antigen-antibody complexes were isolated by affinity absorption to protein G-Sepharose beads and subsequent elution with an SDS-containing buffer. After gel electrophoresis and transfer onto nitrocellulose membrane, the immunoprecipitated proteins were probed with a rabbit polyclonal antibody against RT (Fig.6 A) then, after dehybridation, with a mouse mAb against NCp7 (Fig. 6 B). Significative amounts of NCp7 were retrieved in both cases (Fig. 6 B,lanes 1–2), whereas no nonspecific binding on the beads was observed (Fig. 6 B, lane 3). Moreover, when using HH3 mAb directed against the C-terminal part of NCp7, both subunits, but principally p66, co-immunoprecipitated (Fig.6 A, lane 1), whereas in the case of 2B10 recognizing the first zinc finger of NCp7, essentially p51 was found (Fig. 6 A, lane 2). The light and heavy chains of immunoprecipitating mAbs were also stained (Fig. 6 B), because these antibodies and the probing secondary anti-NCp7 antibody were both elicited in mice. The role of the nucleocapsid protein NCp7 in HIV-1 viral replication is not as yet well defined. In addition to its involvement in dimerization and packaging of genomic RNA and in virus morphogenesis (2Darlix J.L. Lapadat-Tapolsky M. de Rocquigny H. Roques B.P. J. Mol. Biol. 1995; 254: 523-537Crossref PubMed Scopus (381) Google Scholar), several studies suggested that NCp7 may function as a key element of the reverse transcriptionally active ribonucleoprotein complex. Thus, in vitro, NCp7 was shown (i) to promote annealing of the tRNA3Lys to the primer binding site of HIV-1 genome (16Barat C. Lullien V. Schatz O. Keith G. Nugeyre M.T. Grüninger-Leitch F. Barré-Sinoussi F. Le Grice" @default.
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• W2091216865 title "Evidence of Interactions between the Nucleocapsid Protein NCp7 and the Reverse Transcriptase of HIV-1" @default.
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