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• W2065822908 abstract "The replication process of human immunodeficiency virus requires a number of nucleic acid annealing steps facilitated by the hybridization and helix-destabilizing activities of human immunodeficiency virus nucleocapsid (NC) protein. NC contains two CCHC zinc finger motifs numbered 1 and 2 from the N terminus. The amino acids surrounding the CCHC residues differ between the two zinc fingers. Assays were preformed to investigate the activities of the fingers by determining the effect of mutant and wild-type proteins on annealing of 42-nucleotide RNA and DNA complements. The mutants 1.1 NC and 2.2 NC had duplications of the N- and C-terminal zinc fingers in positions 1 and 2. The mutant 2.1 NC had the native zinc fingers with their positions switched. Annealing assays were completed with unstructured and highly structured oligonucleotide complements. 2.2 NC had a near wild-type level of annealing of unstructured nucleic acids, whereas it was completely unable to stimulate annealing of highly structured nucleic acids. In contrast, 1.1 NC was able to stimulate annealing of both unstructured and structured substrates, but to a lesser degree than the wild-type protein. Results suggest that finger 1 has a greater role in unfolding of strong secondary structures, whereas finger 2 serves an accessory role that leads to a further increase in the rate of annealing. The replication process of human immunodeficiency virus requires a number of nucleic acid annealing steps facilitated by the hybridization and helix-destabilizing activities of human immunodeficiency virus nucleocapsid (NC) protein. NC contains two CCHC zinc finger motifs numbered 1 and 2 from the N terminus. The amino acids surrounding the CCHC residues differ between the two zinc fingers. Assays were preformed to investigate the activities of the fingers by determining the effect of mutant and wild-type proteins on annealing of 42-nucleotide RNA and DNA complements. The mutants 1.1 NC and 2.2 NC had duplications of the N- and C-terminal zinc fingers in positions 1 and 2. The mutant 2.1 NC had the native zinc fingers with their positions switched. Annealing assays were completed with unstructured and highly structured oligonucleotide complements. 2.2 NC had a near wild-type level of annealing of unstructured nucleic acids, whereas it was completely unable to stimulate annealing of highly structured nucleic acids. In contrast, 1.1 NC was able to stimulate annealing of both unstructured and structured substrates, but to a lesser degree than the wild-type protein. Results suggest that finger 1 has a greater role in unfolding of strong secondary structures, whereas finger 2 serves an accessory role that leads to a further increase in the rate of annealing. Human immunodeficiency virus (HIV) 1The abbreviations used are: HIV, human immunodeficiency virus; NC, nucleocapsid; MMLV, Moloney murine leukemia virus; FAM, 5′-fluorescein-6-carboxamidohexyl; DABCYL, 4-[[(4-dimethylamino)-phenyl]-azo]benzenesulfonicamino.1The abbreviations used are: HIV, human immunodeficiency virus; NC, nucleocapsid; MMLV, Moloney murine leukemia virus; FAM, 5′-fluorescein-6-carboxamidohexyl; DABCYL, 4-[[(4-dimethylamino)-phenyl]-azo]benzenesulfonicamino. is a member of the family Retroviridae and contains a dimeric single-stranded RNA genome. Within the virion, the genome is coated by nucleocapsid (NC) protein (1Rein A. Henderson L.E. Levin J.G. Trends Biochem. Sci. 1998; 23: 297-301Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 2Darlix J.L. Lapadat-Tapolsky M. de Rocquigny H. Roques B.P. J. Mol. Biol. 1995; 254: 523-537Crossref PubMed Scopus (379) Google Scholar). NC is a basic protein containing 55 amino acids with 15 arginine and lysine residues. This protein contains two CX 2CX 4HX 4C (where X is a variable amino acid) zinc finger motifs that each coordinate a zinc ion (3Fitzgerald D.W. Coleman J.E. Biochemistry. 1991; 30: 5195-5201Crossref PubMed Scopus (53) Google Scholar, 4South T.L. Blake P.R. Sowder III, R.C. Arthur L.O. Henderson L.E. Summers M.F. Biochemistry. 1990; 29: 7786-7789Crossref PubMed Scopus (112) Google Scholar, 5South T.L. Blake P.R. Hare D.R. Summers M.F. Biochemistry. 1991; 30: 6342-6349Crossref PubMed Scopus (98) Google Scholar, 6Summers M.F. South T.L. Kim B. Hare D.R. Biochemistry. 1990; 29: 329-340Crossref PubMed Scopus (261) Google Scholar, 7Berg J.M. Science. 1986; 232: 485-487Crossref PubMed Scopus (720) Google Scholar, 8Chance M.R. Sagi I. Wirt M.D. Frisbie S.M. Scheuring E. Chen E. Bess Jr., J.W. Henderson L.E. Arthur L.O. South T.L. Perez-Alvarado G. Summers M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10041-10045Crossref PubMed Scopus (49) Google Scholar). All known orthoretroviruses contain one or two zinc fingers in their NC proteins (9Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 51Google Scholar). A small basic linker connects the zinc fingers of HIV-1 NC. NMR spectroscopy has shown that the N- and C-terminal tails, as well as the linker region, have a flexible structure (4South T.L. Blake P.R. Sowder III, R.C. Arthur L.O. Henderson L.E. Summers M.F. Biochemistry. 1990; 29: 7786-7789Crossref PubMed Scopus (112) Google Scholar, 10Omichinski J.G. Clore G.M. Sakaguchi K. Appella E. Gronenborn A.M. FEBS Lett. 1991; 292: 25-30Crossref PubMed Scopus (88) Google Scholar, 11Morellet N. Jullian N. de Rocquigny H. Maigret B. Darlix J.L. Roques B.P. EMBO J. 1992; 11: 3059-3065Crossref PubMed Scopus (216) Google Scholar, 12Morellet N. de Rocquigny H. Mely Y. Jullian N. Demene H. Ottmann M. Gerard D. Darlix J.L. Fournie-Zaluski M.C. Roques B.P. J. Mol. Biol. 1994; 235: 287-301Crossref PubMed Scopus (136) Google Scholar, 13Summers M.F. Henderson L.E. Chance M.R. Bess Jr., J.W. South T.L. Blake P.R. Sagi I. Perez-Alvarado G. Sowder III, R.C. Hare D.R. Arthur L.O. Protein Sci. 1992; 1: 563-574Crossref PubMed Scopus (283) Google Scholar). HIV-1 NC is encoded in the gag region of the genome and is a product of proteolytic processing of the Gag precursor protein (14Henderson L.E. Sowder R.C. Copeland T.D. Oroszlan S. Benveniste R.E. J. Med. Primatol. 1990; 19: 411-419Crossref PubMed Google Scholar).HIV-1 NC most often binds to nucleic acids nonspecifically, with an occluded binding site of about seven nucleotides (15You J.C. McHenry C.S. J. Biol. Chem. 1993; 268: 16519-16527Abstract Full Text PDF PubMed Google Scholar, 16Khan R. Giedroc D.P. J. Biol. Chem. 1994; 269: 22538-22546Abstract Full Text PDF PubMed Google Scholar, 17Karpel R.L. Henderson L.E. Oroszlan S. J. Biol. Chem. 1987; 262: 4961-4967Abstract Full Text PDF PubMed Google Scholar). This protein coats the genome within the virion, with the ability to protect the genome against nucleases (17Karpel R.L. Henderson L.E. Oroszlan S. J. Biol. Chem. 1987; 262: 4961-4967Abstract Full Text PDF PubMed Google Scholar, 18Lapadat-Tapolsky M. de Rocquigny H. Van Gent D. Roques B. Plasterk R. Darlix J.L. Nucleic Acids Res. 1993; 21: 831-839Crossref PubMed Scopus (166) Google Scholar). Although it binds the complete genome nonspecifically, in vitro studies have shown an increased affinity for TG and UG repeat sequences in DNA and RNA, respectively (19Vuilleumier C. Bombarda E. Morellet N. Gerard D. Roques B.P. Mely Y. Biochemistry. 1999; 38: 16816-16825Crossref PubMed Scopus (127) Google Scholar, 20Fisher R.J. Rein A. Fivash M. Urbaneja M.A. Casas-Finet J.R. Medaglia M. Henderson L.E. J. Virol. 1998; 72: 1902-1909Crossref PubMed Google Scholar). Also, NMR studies have shown specific interactions between NC residues with stem-loops 2 and 3 of the viral packaging signal (Ψ) (21Amarasinghe G.K. De Guzman R.N. Turner R.B. Chancellor K.J. Wu Z.R. Summers M.F. J. Mol. Biol. 2000; 301: 491-511Crossref PubMed Scopus (308) Google Scholar, 22Amarasinghe G.K. De Guzman R.N. Turner R.B. Summers M.F. J. Mol. Biol. 2000; 299: 145-156Crossref PubMed Scopus (93) Google Scholar, 23De Guzman R.N. Wu Z.R. Stalling C.C. Pappalardo L. Borer P.N. Summers M.F. Science. 1998; 279: 384-388Crossref PubMed Scopus (602) Google Scholar, 24De Guzman R.N. Turner R.B. Summers M.F. Biopolymers. 1998; 48: 181-195Crossref PubMed Scopus (56) Google Scholar). Upon binding to these stem-loops, the N-terminal basic domain of NC forms a 310-helix that interacts with each stem, whereas the C-terminal zinc finger associates with the loop region of the stem-loop (21Amarasinghe G.K. De Guzman R.N. Turner R.B. Chancellor K.J. Wu Z.R. Summers M.F. J. Mol. Biol. 2000; 301: 491-511Crossref PubMed Scopus (308) Google Scholar, 23De Guzman R.N. Wu Z.R. Stalling C.C. Pappalardo L. Borer P.N. Summers M.F. Science. 1998; 279: 384-388Crossref PubMed Scopus (602) Google Scholar).HIV-1 NC involvement has been implicated in many processes in the viral life cycle. Recent studies have shown that NC may play a role in integration of the proviral DNA (18Lapadat-Tapolsky M. de Rocquigny H. Van Gent D. Roques B. Plasterk R. Darlix J.L. Nucleic Acids Res. 1993; 21: 831-839Crossref PubMed Scopus (166) Google Scholar, 25Carteau S. Batson S.C. Poljak L. Mouscadet J.F. de Rocquigny H. Darlix J.L. Roques B.P. Kas E. Auclair C. J. Virol. 1997; 71: 6225-6229Crossref PubMed Google Scholar, 26Carteau S. Gorelick R.J. Bushman F.D. J. Virol. 1999; 73: 6670-6679Crossref PubMed Google Scholar, 27Buckman J.S. Bosche W.J. Gorelick R.J. J. Virol. 2003; 77: 1469-1480Crossref PubMed Scopus (128) Google Scholar). Also, NC sequences in the Gag precursor are a necessity for packaging of the RNA genome (28Gorelick R.J. Chabot D.J. Rein A. Henderson L.E. Arthur L.O. J. Virol. 1993; 67: 4027-4036Crossref PubMed Google Scholar, 29Gorelick R.J. Gagliardi T.D. Bosche W.J. Wiltrout T.A. Coren L.V. Chabot D.J. Lifson J.D. Henderson L.E. Arthur L.O. Virology. 1999; 256: 92-104Crossref PubMed Scopus (121) Google Scholar, 30Poon D.T. Wu J. Aldovini A. J. Virol. 1996; 70: 6607-6616Crossref PubMed Google Scholar) and the maturation of the genomic RNA dimer in the virion core (31Shehu-Xhilaga M. Kraeusslich H.G. Pettit S. Swanstrom R. Lee J.Y. Marshall J.A. Crowe S.M. Mak J. J. Virol. 2001; 75: 9156-9164Crossref PubMed Scopus (70) Google Scholar, 32Feng Y.X. Copeland T.D. Henderson L.E. Gorelick R.J. Bosche W.J. Levin J.G. Rein A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7577-7581Crossref PubMed Scopus (153) Google Scholar). In vivo studies have specifically linked packaging activity to the zinc finger regions of the Gag (NC) precursor (33Tanchou V. Decimo D. Pechoux C. Lener D. Rogemond V. Berthoux L. Ottmann M. Darlix J.L. J. Virol. 1998; 72: 4442-4447Crossref PubMed Google Scholar, 34Rein A. Harvin D.P. Mirro J. Ernst S.M. Gorelick R.J. J. Virol. 1994; 68: 6124-6129Crossref PubMed Google Scholar). In vitro studies have indicated that NC may have important roles in reverse transcription as well. HIV-1 NC has been shown to enhance annealing of the tRNA primer to the primer-binding site to initiate reverse transcription (35Brule F. Marquet R. Rong L. Wainberg M.A. Roques B.P. Le Grice S.F. Ehresmann B. Ehresmann C. RNA (N. Y.). 2002; 8: 8-15Crossref PubMed Scopus (38) Google Scholar, 36Feng Y.X. Campbell S. Harvin D. Ehresmann B. Ehresmann C. Rein A. J. Virol. 1999; 73: 4251-4256Crossref PubMed Google Scholar, 37Khan R. Giedroc D.P. J. Biol. Chem. 1992; 267: 6689-6695Abstract Full Text PDF PubMed Google Scholar, 38Rong L. Liang C. Hsu M. Kleiman L. Petitjean P. de Rocquigny H. Roques B.P. Wainberg M.A. J. Virol. 1998; 72: 9353-9358Crossref PubMed Google Scholar); to stimulate minus, plus, and internal transfer (39Allain B. Lapadat-Tapolsky M. Berlioz C. Darlix J.L. EMBO J. 1994; 13: 973-981Crossref PubMed Scopus (157) Google Scholar, 40DeStefano J.J. Arch. Virol. 1995; 140: 1775-1789Crossref PubMed Scopus (46) Google Scholar, 41Guo J. Henderson L.E. Bess J. Kane B. Levin J.G. J. Virol. 1997; 71: 5178-5188Crossref PubMed Google Scholar, 42Kim J.K. Palaniappan C. Wu W. Fay P.J. Bambara R.A. J. Biol. Chem. 1997; 272: 16769-16777Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 43Peliska J.A. Balasubramanian S. Giedroc D.P. Benkovic S.J. Biochemistry. 1994; 33: 13817-13823Crossref PubMed Scopus (161) Google Scholar, 44You J.C. McHenry C.S. J. Biol. Chem. 1994; 269: 31491-31495Abstract Full Text PDF PubMed Google Scholar, 45Raja A. DeStefano J.J. Biochemistry. 1999; 38: 5178-5184Crossref PubMed Scopus (25) Google Scholar); and to have an effect on the processivity of reverse transcriptase in DNA synthesis (46Rodriguez-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, 47Ji X. Klarmann G.J. Preston B.D. Biochemistry. 1996; 35: 132-143Crossref PubMed Scopus (116) Google Scholar, 48Lener D. Tanchou V. Roques B.P. Le Grice S.F. Darlix J.L. J. Biol. Chem. 1998; 273: 33781-33786Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar).HIV-1 NC has also been shown to possess chaperone activity (1Rein A. Henderson L.E. Levin J.G. Trends Biochem. Sci. 1998; 23: 297-301Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 18Lapadat-Tapolsky M. de Rocquigny H. Van Gent D. Roques B. Plasterk R. Darlix J.L. Nucleic Acids Res. 1993; 21: 831-839Crossref PubMed Scopus (166) Google Scholar, 49Lapadat-Tapolsky M. Pernelle C. Borie C. Darlix J.L. Nucleic Acids Res. 1995; 23: 2434-2441Crossref PubMed Scopus (162) Google Scholar, 50Dib-Hajj F. Khan R. Giedroc D.P. Protein Sci. 1993; 2: 231-243Crossref PubMed Scopus (95) Google Scholar, 51Tsuchihashi Z. Brown P.O. J. Virol. 1994; 68: 5863-5870Crossref PubMed Google Scholar, 52Williams M.C. Rouzina I. Wenner J.R. Gorelick R.J. Musier-Forsyth K. Bloomfield V.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6121-6126Crossref PubMed Scopus (154) Google Scholar, 53Urbaneja M.A. Wu M. Casas-Finet J.R. Karpel R.L. J. Mol. Biol. 2002; 318: 749-764Crossref PubMed Scopus (91) Google Scholar), meaning it can aid in the unfolding of nucleic acid structures to enhance the annealing of more thermodynamically favorable structures (containing more base pairs) (54Herschlag D. J. Biol. Chem. 1995; 270: 20871-20874Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar). This chaperone activity aids in tRNA/primer-binding site annealing, annealing of RNA and DNA in strand transfer, and genome dimerization and maturation. It is not clear if there are particular regions of NC that are responsible for chaperone activity or various components (helix destabilization (unwinding) or hybridization of complements) of this chaperone activity. A number of investigators have completed annealing assays with mutant NC proteins to determine what residues are necessary for NC chaperone capability. Studies examining the effect of Moloney murine leukemia virus (MMLV) NC mutants on the annealing of tRNAPro to MMLV RNA, as well as the dimerization of the genomic RNA segments, showed that the basic regions of NC are necessary for enhancement (55Prats A.C. Housset V. de Billy G. Cornille F. Prats H. Roques B. Darlix J.L. Nucleic Acids Res. 1991; 19: 3533-3541Crossref PubMed Scopus (74) Google Scholar, 56de Rocquigny H. Ficheux D. Gabus C. Allain B. Fournie-Zaluski M.C. Darlix J.L. Roques B.P. Nucleic Acids Res. 1993; 21: 823-829Crossref PubMed Scopus (38) Google Scholar). These studies indicated that the single zinc finger of MMLV NC could be removed and that chaperone activity was retained. Similar experiments were completed with genomic RNA sequences and NC derived from HIV. RNA dimerization and tRNA3Lys binding were observed in the presence of HIV-1 NC mutants containing only amino acid sequences exterior to the zinc fingers (49Lapadat-Tapolsky M. Pernelle C. Borie C. Darlix J.L. Nucleic Acids Res. 1995; 23: 2434-2441Crossref PubMed Scopus (162) Google Scholar, 57Takahashi K. Baba S. Koyanagi Y. Yamamoto N. Takaku H. Kawai G. J. Biol. Chem. 2001; 276: 31274-31278Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 58Hsu M. Rong L. de Rocquigny H. Roques B.P. Wainberg M.A. Nucleic Acids Res. 2000; 28: 1724-1729Crossref PubMed Scopus (20) Google Scholar, 59de Rocquigny H. Gabus C. Vincent A. Fournie-Zaluski M.C. Roques B. Darlix J.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6472-6476Crossref PubMed Scopus (279) Google Scholar). Mutants with zinc fingers replaced by glycine-glycine linkers were able to enhance annealing, although the concentration of these proteins was often higher than used with wild-type NC. In vitro experiments have also been completed with ribozymes that will cleave RNA sequences upon annealing to them (60Muller G. Strack B. Dannull J. Sproat B.S. Surovoy A. Jung G. Moelling K. J. Mol. Biol. 1994; 242: 422-429Crossref PubMed Scopus (42) Google Scholar, 61Tsuchihashi Z. Khosla M. Herschlag D. Science. 1993; 262: 99-102Crossref PubMed Scopus (190) Google Scholar). Enhanced RNA cleavage was observed in the presence of wild-type NC as well as mutants containing the conserved basic residues with deleted zinc fingers (60Muller G. Strack B. Dannull J. Sproat B.S. Surovoy A. Jung G. Moelling K. J. Mol. Biol. 1994; 242: 422-429Crossref PubMed Scopus (42) Google Scholar). These reports indicate that the basic residues of NC are an absolute requirement for chaperone activity (56de Rocquigny H. Ficheux D. Gabus C. Allain B. Fournie-Zaluski M.C. Darlix J.L. Roques B.P. Nucleic Acids Res. 1993; 21: 823-829Crossref PubMed Scopus (38) Google Scholar, 59de Rocquigny H. Gabus C. Vincent A. Fournie-Zaluski M.C. Roques B. Darlix J.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6472-6476Crossref PubMed Scopus (279) Google Scholar, 60Muller G. Strack B. Dannull J. Sproat B.S. Surovoy A. Jung G. Moelling K. J. Mol. Biol. 1994; 242: 422-429Crossref PubMed Scopus (42) Google Scholar, 62Urbaneja M.A. Kane B.P. Johnson D.G. Gorelick R.J. Henderson L.E. Casas-Finet J.R. J. Mol. Biol. 1999; 287: 59-75Crossref PubMed Scopus (79) Google Scholar).Other annealing experiments have also been completed with NC proteins that retained the zinc fingers, but that contained mutated residues within these regions. Guo et al. (63Guo J. Wu T. Kane B.F. Johnson D.G. Henderson L.E. Gorelick R.J. Levin J.G. J. Virol. 2002; 76: 4370-4378Crossref PubMed Scopus (92) Google Scholar) completed annealing assays using sequences from the trans-activation response region of the HIV-1 genome. They used three types of proteins with mutations that alter either the structure of the zinc finger or the residues within the zinc finger that are not involved in zinc binding. Optimal annealing activity was observed only when the N-terminal zinc finger was not mutated. The effects of mutations to the C-terminal zinc finger were not as pronounced, although these results varied with the type of mutant constructed. The results are supported by experiments that tested the effect of altering the zinc finger structure on tRNA3Lys annealing to the primer-binding site (64Remy 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). In this case, the zinc fingers were shown to be necessary for the annealing reaction. Also, annealing assays completed with the nucleic acid sequences from the regions of (–)- and (+)-strand transfer in the presence of NC mutants unable to bind zinc showed that the requirement for the zinc finger structure differed depending on the nucleic acid sequence being used (65Guo J. Wu T. Anderson J. Kane B.F. Johnson D.G. Gorelick R.J. Henderson L.E. Levin J.G. J. Virol. 2000; 74: 8980-8988Crossref PubMed Scopus (177) Google Scholar). Last, single DNA molecule stretching experiments have been completed with NC to examine its ability to destabilize the helix and to aid in transition to a coil structure. HIV-1 NC mutants were used in these experiments, and it was observed that the N-terminal finger of HIV-1 NC must be in the native position for optimal chaperone activity (52Williams M.C. Rouzina I. Wenner J.R. Gorelick R.J. Musier-Forsyth K. Bloomfield V.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6121-6126Crossref PubMed Scopus (154) Google Scholar, 66Williams M.C. Gorelick R.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8614-8619Crossref PubMed Scopus (96) Google Scholar).The study presented here investigates how NC-enhanced nucleic acid annealing differs for unstructured and structured nucleic acids. NC was shown to increase the annealing of both types of nucleic acids, indicating that it stimulates both the unfolding of structured nucleic acids and the direct hybridization of complements. NC zinc finger mutants were also used in annealing assays, which provided additional insight into the potential role of zinc fingers in annealing. The N-terminal zinc finger was shown to be necessary for the annealing of sequences with a high degree of secondary structure, whereas the C-terminal zinc finger was shown to be required for maximal annealing activity. Overall, the results suggest that the N-terminal finger is more important in unwinding secondary structures, whereas the C-terminal finger plays an accessory role.EXPERIMENTAL PROCEDURESMaterials—RNA oligonucleotides were purchased from Dharmicon Research, Inc. DNA oligonucleotides were purchased from Integrated DNA Technologies and Invitrogen. T4 polynucleotide kinase, Klenow polymerase, and T4 RNA ligase were obtained from New England Biolabs Inc. SP6 polymerase, alkaline phosphatase, NTPs, dNTPs, and RNase-free DNase I were obtained from Roche Applied Science. Radiolabeled compounds were purchased from PerkinElmer Life Sciences. All other chemicals were from Fisher or Sigma.Preparation of Wild-type and Mutant NC Proteins—HIV-1 NC from the HIV-1 AIDS-associated retrovirus strain was prepared as described (15You J.C. McHenry C.S. J. Biol. Chem. 1993; 268: 16519-16527Abstract Full Text PDF PubMed Google Scholar). Wild-type and mutant NC proteins from the HIV-1 NL4-3 strain were prepared as explained previously (26Carteau S. Gorelick R.J. Bushman F.D. J. Virol. 1999; 73: 6670-6679Crossref PubMed Google Scholar). There are four differing amino acids between the NC proteins derived from the two HIV strains. NC from the AIDS-associated retrovirus strain contains two arginine residues in the C-terminal zinc finger that are substituted with lysine residues in NC from the NL4-3 strain. Aliquots of HIV-1 NC were prepared and stored in 50 mm Tris-HCl (pH 7.5), 10% glycerol, and 5 mm 2-mercaptoethanol at –80 °C. Fresh aliquots were used for each experiment.Preparation of Oligonucleotides—RNA oligonucleotide 0.0rna was purchased from Dharmicon Research, Inc., and DNA oligonucleotide 0.0dna was from Invitrogen. The most highly structured pair of oligonucleotides, 21.7rna and 21.7dna, were purchased from Integrated DNA Technologies. The RNA contained a 5′-fluorescein-6-carboxamidohexyl (FAM) end label, whereas the DNA contained a 4-[[(4-dimethylamino)phenyl]-azo]benzenesulfonicamino (DABCYL) group. Oligonucleotides 7.5rna and 16.3rna were transcribed from DNA oligonucleotide pairs. One DNA strand of each pair was 61 nucleotides long and contained the sequence for the SP6 promoter at the 5′-end, followed by the DNA sequence corresponding to the desired RNA. Twenty pmol of the 61-mer was combined with 40 pmol of a complementary 42-mer DNA in hybridization buffer (50 mm Tris-HCl (pH 8.0), 1mm dithiothreitol, and 80 mm KCl). The hybrid reaction was heated to 65 °C for 5 min and then cooled slowly to room temperature. The 5′-overhang was filled in using Klenow polymerase at 37 °C. The hybrid was incubated for 1 h with Klenow polymerase and 200 μm dNTPs. The double-stranded DNA product was extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with ethanol. SP6 polymerase was then used to transcribe the 42-nucleotide RNA product. DNA was digested with DNase I for 10 min at 37 °C. The reaction was stopped by adding an equal volume of 2× formamide dye. The RNA was heated to 90 °C for 3 min and then subjected to gel electrophoresis on an RNase-free 10% denaturing polyacrylamide gel. RNA was excised, eluted in formamide elution buffer (80% formamide, 400 mm NaCl, 1 mm EDTA, and 40 mm Tris-HCl (pH 7.0)), and precipitated with ethanol. The 5′-end of the RNA was dephosphorylated using calf intestinal alkaline phosphatase. Seventy-five pmol of RNA was incubated with calf intestinal alkaline phosphatase for 1 h at 37 °C. The dephosphorylated RNA was extracted with phenol/chloroform/isoamyl alcohol (25: 24:1) and precipitated with ethanol. All of the aforementioned oligonucleotides were resuspended in 30 μl of water and quantitated spectrophotometrically.RNA End Labeling—Oligonucleotides 0.0rna, 7.5rna, and 16.3rna were labeled at the 5′-end with [γ-32P]ATP. Fifty pmol of dephosphorylated RNA was end-labeled using T4 polynucleotide kinase. The 5′-end of the most highly structured sequence, 21.7rna, was connected to a FAM molecule. Therefore, this RNA was 3′-end-labeled with [5′-32P]cytidine 3′,5′-bisphosphate as described. 2J. W. Brown, unpublished method. All labeled RNAs were purified on a 10% polyacrylamide gel, eluted, and precipitated as described above. RNAs were resuspended in 70 μl of TE buffer (10 mm Tris-HCl (pH 8) and 1 mm EDTA (pH 8)) and quantitated spectrophotometrically.RNA/DNA Annealing Assay— 32P-5′-end-labeled RNA and the complementary DNA were diluted in TE buffer, separately heated to 90 °C for 3 min, and then transferred to ice for 5 min. The RNA (5 nm final oligonucleotide concentration and 0.21 μm nucleotide concentration) was then preincubated at 37 °C in the presence or absence of mutant or wild-type HIV-1 NC (2 μm final concentration) for 2 min. Complementary DNA (10 nm final concentration and 0.42 μm nucleotide concentration) was separately preincubated at 37 °C for 2 min in the presence or absence of HIV-1 NC (2 μm final concentration). To start the reactions, 17 μl of DNA solution was added to 90 μl of reaction mixture containing the RNA. Final reaction concentrations were 50 mm Tris-HCl (pH 8.0), 0.1 mm EDTA, 1 mm dithiothreitol, 6 mm MgCl2,80mm KCl, and 100 μm ZnCl2. Aliquots of 15 μl were removed at specific time points (as indicated) and added to 7.5 μl of stop solution (0.25% bromphenol blue, 20% glycerol, 20 mm EDTA (pH 8), 0.2% SDS, and 0.4 mg/ml yeast tRNA) (51Tsuchihashi Z. Brown P.O. J. Virol. 1994; 68: 5863-5870Crossref PubMed Google Scholar). All reactions were incubated in stop solution at 37 °C for 1 min before being transferred to ice. Reactions were then subjected to electrophoresis on 12 or 15% native polyacrylamide gels. Gels were dried and subjected to autoradiography (67Sambrook J. Russell D.W. Irwin N. Janssen K.A. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar) or phosphoimager analysis using a Bio-Rad GS-525 phosphoimager. Percent annealing was determined by dividing the amount of annealed product (A) in each lane by the total RNA (annealed and single-stranded (S)) in each lane and multiplying by 100 (% annealed = A/(A + S) × 100).Annealing Detected by Fluorescence Resonance Energy Transfer—As was previously noted, 21.7rna was purchased with a FAM molecule on its 5′-end. The complementary DNA was purchased with a 3′-DABCYL group. Annealing assays were completed at 25 °C using a Fluoromax-2 spectrofluorometer (Jobin Yvon Instruments S. A., Inc.). The RNA and DNA were separately incubated in the presence or absence of wild-type NC, 1.1 NC, or 2.2 NC. The reactions were started by mixing 10.5 μl of the DNA/NC solution and 59.5 μl of the RNA/NC solution. The final concentrations of the RNA and DNA were 5 and 10 nm, respectively (0.63 μm total nucleotide concentration). NC was used at a final concentration of 2 μm, and the final concentrations of the reagents in the buffer were as follows: 50 mm Tris-HCl (pH 8.0), 0.1 mm EDTA, 1 mm dithiothreitol, 6 mm MgCl2, 80 mm KCl, and 100 μm ZnCl2. The excitation wavelength was 494 nm with a bandwidth of 1 nm. The emission bandwidth was 5 nm, and the spectrum was observed from 508 to 570 nm. The emission spectrum was taken at 0, 1, 2, 4, 8, and 16 min. The intensity of the emission peaked at 517 nm. The intensity ratio (I r) was determined by dividing the peak intensity at a given time (I t) by the peak intensity at time 0 (I 0) (I r = I t/I 0). Three experiments were completed for each type of NC, and an average intensity ratio was determined and plotted versus time.RESULTSStructure of Substrate RNAs—The RNAs used in these experiments each contained 42 nucleotides. The structures are shown in Fig. 1. These structures were determined using both RNAdraw and mfold (68Zuker M. Stiegler P. Nucleic Acids Res. 1981; 9: 133-148Crossref PubMed Scopus (2589) Google Scholar, 69Matzura O. Wennborg A. Comput. Appl. Biosci. 1996; 12: 247-249PubMed Google Scholar). These structures were confirmed using RNase A and RNase T1 mapping (data not shown). The Gibbs free energy for unfolding is shown next to the structure. Nucleic acids were designed such that they would contain the same number of nucleotides, but have an increasing strength of secondary structures. Structures were chosen that did not have a high degree of GU (or GT in the complementary DNA) repeats due to reports indicating that NC may preferentially bind to these sequences (19Vuilleumier C. Bombarda E. Morellet N. Gerard D. Roques B.P. Mely Y. Biochemistry. 1999; 38: 16816-16825Crossref PubMed Scopus (127) Google Scholar, 20Fisher R.J. Rein A. Fivash M. Urbaneja M.A. Casas-Finet J.R. Medaglia M. Henderson L.E. J. Virol. 1998; 72: 1902-1909Crossref PubMed Google Scholar).HIV-1 NC-enhanced Annealing of Structured and Unstructured RNAs—Annealing assays were completed to examine how HIV-1 NC affects the an" @default.
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• W2065822908 title "Differing Roles of the N- and C-terminal Zinc Fingers in Human Immunodeficiency Virus Nucleocapsid Protein-enhanced Nucleic Acid Annealing" @default.
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