HIV-1 nucleocapsid protein activates transient melting of least stable parts of the secondary structure of TAR and its complementary sequence.

The nucleocapsid protein NCp7 of HIV-1 possesses a nucleic acid chaperone activity that is critical in minus and plus strand transfer during reverse transcription. The minus strand transfer notably relies on the ability of NCp7 to destabilize the stable stem with five contiguous, double-stranded segments of both the TAR sequence at the 3' end of the viral genome and the complementary sequence, cTAR, in minus strong-stop DNA. In order to examine the nature and the extent of NCp7 destabilizing activity, we investigated, by absorbance and fluorescence spectroscopy, the interaction of TAR and cTAR with a (12-55)NCp7 peptide containing the zinc-finger motifs but lacking the ability to aggregate the oligonucleotides. The absorbance changes in the UV band of cTAR show that seven to eight base-pairs, on average, are melted per oligonucleotide at a ratio of one peptide to 7.5 nucleotides. In contrast, the melting of TAR does not exceed an average of one base-pair per oligonucleotide. This may be linked to the greater stability of TAR, since a strong correlation between NCp7 destabilizing effect and oligonucleotide stability was observed. The effect of (12-55)NCp7 on the stem terminus was investigated by using a cTAR molecule doubly labeled at the 3' and 5' ends by a donor/acceptor couple. In the absence of the peptide, about 80 % of the oligonucleotides are in a dark non-fluorescent state, having a close proximity of the two dyes. The remaining 20 % are distributed between three fluorescent species, having either the terminal segment, the two terminal segments or all segments of the stem melted. This is in line with a fraying mechanism wherein the stem terminus fluctuates rapidly between open and closed states. Addition of (12-55)NCp7 shifts the equilibrium toward the open species, suggesting that NC enhances fraying of the stem terminus. Taken together, our data suggest that NCp7 activates the transient opening of base-pairs in the least stable parts of the stem. Also, this activity of NCp7 was found to be dependent on the zinc-finger motifs, since no melting was observed with a fingerless NCp7 peptide.

[1]  K. Musier-Forsyth,et al.  HIV-1 nucleocapsid protein zinc finger structures induce tRNA(Lys,3) structural changes but are not critical for primer/template annealing. , 2001, Journal of molecular biology.

[2]  Serena Bernacchi,et al.  Exciton interaction in molecular beacons: a sensitive sensor for short range modifications of the nucleic acid structure , 2001, Nucleic Acids Res..

[3]  K. Musier-Forsyth,et al.  Mechanism for nucleic acid chaperone activity of HIV-1 nucleocapsid protein revealed by single molecule stretching , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[4]  B. Roques,et al.  Heteronuclear NMR studies of the interaction of tRNA3Lys with HIV-1 nucleocapsid protein , 2001 .

[5]  S. Hughes,et al.  Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Can Prevent Self-Priming of Minus-Strand Strong Stop DNA by Promoting the Annealing of Short Oligonucleotides to Hairpin Sequences , 2000, Journal of Virology.

[6]  Jianhui Guo,et al.  Zinc Finger Structures in the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Facilitate Efficient Minus- and Plus-Strand Transfer , 2000, Journal of Virology.

[7]  J G Levin,et al.  A mechanism for plus-strand transfer enhancement by the HIV-1 nucleocapsid protein during reverse transcription. , 2000, Biochemistry.

[8]  Liming Ying,et al.  Ratiometric Analysis of Single-Molecule Fluorescence Resonance Energy Transfer Using Logical Combinations of Threshold Criteria: A Study of 12-mer DNA , 2000 .

[9]  M. Wainberg,et al.  The effect of mutations in the HIV-1 nucleocapsid protein on strand transfer in cell-free reverse transcription reactions. , 2000, Nucleic acids research.

[10]  B. Roques,et al.  Nucleic acid sequence discrimination by the HIV-1 nucleocapsid protein NCp7: a fluorescence study. , 1999, Biochemistry.

[11]  H. Hoffmann,et al.  AGGREGATION OF POLYA-HIV-1 NUCLEOCAPSID PROTEIN NCP7 COMPLEXES AND PROPERTIES OF THE AGGREGATES , 1999 .

[12]  J. Bess,et al.  Molecular Requirements for Human Immunodeficiency Virus Type 1 Plus-Strand Transfer: Analysis in Reconstituted and Endogenous Reverse Transcription Systems , 1999, Journal of Virology.

[13]  C. Ehresmann,et al.  The Human Immunodeficiency Virus Type 1 Gag Polyprotein Has Nucleic Acid Chaperone Activity: Possible Role in Dimerization of Genomic RNA and Placement of tRNA on the Primer Binding Site , 1999, Journal of Virology.

[14]  J. Lifson,et al.  Strict conservation of the retroviral nucleocapsid protein zinc finger is strongly influenced by its role in viral infection processes: characterization of HIV-1 particles containing mutant nucleocapsid zinc-coordinating sequences. , 1999, Virology.

[15]  S. L. Le Grice,et al.  Role of Post-transcriptional Modifications of Primer tRNALys,3 in the Fidelity and Efficacy of Plus Strand DNA Transfer during HIV-1 Reverse Transcription* , 1999, The Journal of Biological Chemistry.

[16]  P. Barbara,et al.  Intra-tRNA distance measurements for nucleocapsid proteindependent tRNA unwinding during priming of HIV reverse transcription. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[17]  A. Rein,et al.  Nucleic-acid-chaperone activity of retroviral nucleocapsid proteins: significance for viral replication. , 1998, Trends in biochemical sciences.

[18]  A Libchaber,et al.  Kinetics of conformational fluctuations in DNA hairpin-loops. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[19]  C. Bustamante,et al.  Polymer chain statistics and conformational analysis of DNA molecules with bends or sections of different flexibility. , 1998, Journal of molecular biology.

[20]  C. Péchoux,et al.  Role of the N-Terminal Zinc Finger of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein in Virus Structure and Replication , 1998, Journal of Virology.

[21]  J. Casas-Finet,et al.  Sequence-Specific Binding of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein to Short Oligonucleotides , 1998, Journal of Virology.

[22]  B. Roques,et al.  The Annealing of tRNA3 Lys to Human Immunodeficiency Virus Type 1 Primer Binding Site Is Critically Dependent on the NCp7 Zinc Fingers Structure* , 1998, The Journal of Biological Chemistry.

[23]  J. SantaLucia,et al.  A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[24]  R. Rigler,et al.  Conformational fluctuations in single DNA molecules. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[25]  R. Bambara,et al.  Evidence for a Unique Mechanism of Strand Transfer from the Transactivation Response Region of HIV-1* , 1997, The Journal of Biological Chemistry.

[26]  J G Levin,et al.  Human immunodeficiency virus type 1 nucleocapsid protein promotes efficient strand transfer and specific viral DNA synthesis by inhibiting TAR-dependent self-priming from minus-strand strong-stop DNA , 1997, Journal of virology.

[27]  C. Gabus,et al.  Possible roles of HIV-1 nucleocapsid protein in the specificity of proviral DNA synthesis and in its variability. , 1997, Journal of molecular biology.

[28]  Jerker Widengren,et al.  Fast interactions between Rh6G and dGTP in water studied by fluorescence correlation spectroscopy , 1997 .

[29]  B. Roques,et al.  Ordered aggregation of ribonucleic acids by the human immunodeficiency virus type 1 nucleocapsid protein. , 1997, Biopolymers.

[30]  G. Glick,et al.  The effect of cross-links on the conformational dynamics of duplex DNA. , 1997, Nucleic acids research.

[31]  R. Gorelick,et al.  Human immunodeficiency virus type 1 nucleocapsid protein reduces reverse transcriptase pausing at a secondary structure near the murine leukemia virus polypurine tract , 1996, Journal of virology.

[32]  D. Giedroc,et al.  Interaction of retroviral nucleocapsid proteins with transfer RNAPhe: a lead ribozyme and 1H NMR study. , 1996, Nucleic acids research.

[33]  R. Clegg,et al.  Fluorescence characteristics of 5-carboxytetramethylrhodamine linked covalently to the 5' end of oligonucleotides: multiple conformers of single-stranded and double-stranded dye-DNA complexes. , 1996, Biophysical journal.

[34]  R. Rigler,et al.  Conformational transitions monitored for single molecules in solution. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[35]  C. Bustamante,et al.  Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules , 1996, Science.

[36]  G. Klarmann,et al.  Effect of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein on HIV-1 reverse transcriptase activity in vitro. , 1996, Biochemistry.

[37]  K. Breslauer,et al.  Influence of the oxidatively damaged adduct 8-oxodeoxyguanosine on the conformation, energetics, and thermodynamic stability of a DNA duplex. , 1995, Biochemistry.

[38]  B. Roques,et al.  First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses. , 1995, Journal of molecular biology.

[39]  C. Gabus,et al.  Formation of stable and functional HIV-1 nucleoprotein complexes in vitro. , 1995, Journal of molecular biology.

[40]  D. Herschlag.  RNA Chaperones and the RNA Folding Problem (*) , 1995, The Journal of Biological Chemistry.

[41]  M. Guéron,et al.  Terminal base pairs of oligodeoxynucleotides: imino proton exchange and fraying. , 1995, Biochemistry.

[42]  Mary Lapadat-Tapolsky,et al.  Analysis of the nucleic acid annealing activities of nucleocapsid protein from HIV-1 , 1995, Nucleic Acids Res..

[43]  Z. Tsuchihashi,et al.  Influence of Human Immunodeficiency Virus Nucleocapsid Protein on Synthesis and Strand Transfer by the Reverse Transcriptase in Vitro(*) , 1995, The Journal of Biological Chemistry.

[44]  B. Roques,et al.  Binding of the HIV-1 Nucleocapsid Protein to the Primer tRNA, inVitro, Is Essentially Not Specific (*) , 1995, The Journal of Biological Chemistry.

[45]  C. McHenry,et al.  Human immunodeficiency virus nucleocapsid protein accelerates strand transfer of the terminally redundant sequences involved in reverse transcription. , 1994, The Journal of biological chemistry.

[46]  S. Balasubramanian,et al.  Recombinant HIV-1 nucleocapsid protein accelerates HIV-1 reverse transcriptase catalyzed DNA strand transfer reactions and modulates RNase H activity. , 1994, Biochemistry.

[47]  N. Jullian,et al.  Spatial proximity of the HIV-1 nucleocapsid protein zinc fingers investigated by time-resolved fluorescence and fluorescence resonance energy transfer. , 1994, Biochemistry.

[48]  R. Bischoff,et al.  Negative electrospray ionization mass spectrometry of synthetic and chemically modified oligonucleotides. , 1994, Nucleic acids research.

[49]  P. Brown,et al.  DNA strand exchange and selective DNA annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein , 1994, Journal of virology.

[50]  W. Fu,et al.  Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease-defective virions , 1994, Journal of virology.

[51]  K. Breslauer,et al.  DNA Lesions: A Thermodynamic Perspective , 1994, Annals of the New York Academy of Sciences.

[52]  S. Goff,et al.  Analysis of binding elements in the human immunodeficiency virus type 1 genomic RNA and nucleocapsid protein. , 1994, Virology.

[53]  D. Herschlag,et al.  An RNA chaperone activity of non‐specific RNA binding proteins in hammerhead ribozyme catalysis. , 1994, The EMBO journal.

[54]  J. Darlix,et al.  Transactivation of the minus‐strand DNA transfer by nucleocapsid protein during reverse transcription of the retroviral genome. , 1994, The EMBO journal.

[55]  J. Luban,et al.  Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays , 1993, Journal of virology.

[56]  W. Fu,et al.  Maturation of dimeric viral RNA of Moloney murine leukemia virus , 1993, Journal of virology.

[57]  B. Roques,et al.  Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA. , 1993, Comptes rendus de l'Academie des sciences. Serie III, Sciences de la vie.

[58]  R. Plasterk,et al.  Interactions between HIV-1 nucleocapsid protein and viral DNA may have important functions in the viral life cycle. , 1993, Nucleic acids research.

[59]  C. Ehresmann,et al.  Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains. , 1993, Journal of molecular biology.

[60]  J. Moe,et al.  Kinetics and energetics of base-pair opening in 5'-d(CGCGAATTCGCG)-3' and a substituted dodecamer containing G.T mismatches. , 1992, Biochemistry.

[61]  N. Jullian,et al.  Determination of the structure of the nucleocapsid protein NCp7 from the human immunodeficiency virus type 1 by 1H NMR. , 1992, The EMBO journal.

[62]  M. Chance,et al.  Nucleocapsid zinc fingers detected in retroviruses: EXAFS studies of intact viruses and the solution‐state structure of the nucleocapsid protein from HIV‐1 , 1992, Protein science : a publication of the Protein Society.

[63]  D. Giedroc,et al.  Recombinant human immunodeficiency virus type 1 nucleocapsid (NCp7) protein unwinds tRNA. , 1992, The Journal of biological chemistry.

[64]  H. Issaq,et al.  Tightly bound zinc in human immunodeficiency virus type 1, human T-cell leukemia virus type I, and other retroviruses , 1992, Journal of virology.

[65]  B. Roques,et al.  First large scale chemical synthesis of the 72 amino acid HIV-1 nucleocapsid protein NCp7 in an active form. , 1991, Biochemical and biophysical research communications.

[66]  D. Patel,et al.  Proton exchange and internal motions in two chromomycin dimer-DNA oligomer complexes. , 1991, Biochemistry.

[67]  M. Summers.  Zinc finger motif for single‐stranded nucleic acids? investigations by nuclear magnetic resonance , 1991, Journal of cellular biochemistry.

[68]  F. Barré-Sinoussi,et al.  Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1. , 1990, Journal of molecular biology.

[69]  R. Young,et al.  Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus , 1990, Journal of virology.

[70]  F. Barré-Sinoussi,et al.  HIV‐1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. , 1989, The EMBO journal.

[71]  M. Guéron,et al.  Evidence from base-pair kinetics for two types of adenine tract structures in solution: their relation to DNA curvature. , 1988, Biochemistry.

[72]  J. Brochon,et al.  Analyzing the distribution of decay constants in pulse-fluorimetry using the maximum entropy method. , 1987, Biophysical journal.

[73]  M. Guéron,et al.  A single mode of DNA base-pair opening drives imino proton exchange , 1987, Nature.

[74]  J. Berg,et al.  Potential metal-binding domains in nucleic acid binding proteins. , 1986, Science.

[75]  J. Coffin.  Structure of the retroviral genome , 1982 .

[76]  D. Magde,et al.  Absolute luminescence yield of cresyl violet. A standard for the red , 1979 .

[77]  J. Brochon.  Maximum entropy method of data analysis in time-resolved spectroscopy. , 1994, Methods in enzymology.

[78]  G. Weber,et al.  Determination of the absolute quantum yield of fluorescent solutions , 1957 .