Characterization of active reverse transcriptase and nucleoprotein complexes of the yeast retrotransposon Ty3 in vitro

被引:25
作者
Cristofari, G
Gabus, C
Ficheux, D
Bona, M
Le Grice, SFJ
Darlix, JL
机构
[1] Ecole Normale Super Lyon, INSERM, LaboRetro, Unite Virol Humaine,U412, F-69364 Lyon 07, France
[2] Inst Biol & Chim Prot, F-69367 Lyon, France
[3] NCI, Frederick Canc Res & Dev Ctr, HIV Drug Resistance Program, Div Basic Sci,NIH, Frederick, MD 21702 USA
关键词
D O I
10.1074/jbc.274.51.36643
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively. In vitro binding of HIV-1 NCp7 to viral 5' RNA and primer tRNA(3)(Lys) catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid. Nucleocapsid complex formation functions in viral RNA dimerization and tRNA annealing to the primer binding site (PBS). RT is recruited in these nucleoprotein complexes and synthesizes minus-strand cDNA initiated at the PBS. Recent results on yeast Ty3 have shown that the homologous NCp9 promotes annealing of primer tRNA(i)(Met) to a 5'-3' bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5' PBS (1), To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3 RNA with the 5'-3' PBS, primer tRNA(i)(Met), NCp9, and for the first time, highly purified Ty3 RT. Here we report that Ty3 RT is as active as retroviral HIV-1 or murine leukemia virus RT using a synthetic template-primer system, Moreover, and in contrast to what was found with retroviral RTs, retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming. We also show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT. These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7.
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页码:36643 / 36648
页数:6
相关论文
共 41 条
  • [1] TRANSACTIVATION OF THE MINUS-STRAND DNA TRANSFER BY NUCLEOCAPSID PROTEIN DURING REVERSE TRANSCRIPTION OF THE RETROVIRAL GENOME
    ALLAIN, B
    LAPADATTAPOLSKY, M
    BERLIOZ, C
    DARLIX, JL
    [J]. EMBO JOURNAL, 1994, 13 (04) : 973 - 981
  • [2] Role of post-transcriptional modifications of primer tRNALys,3 in the fidelity and efficacy of plus strand DNA transfer during HIV-1 reverse transcription
    Auxilien, S
    Keith, G
    Le Grice, SFJ
    Darlix, JL
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (07) : 4412 - 4420
  • [3] HIV-1 REVERSE-TRANSCRIPTASE SPECIFICALLY INTERACTS WITH THE ANTICODON DOMAIN OF ITS COGNATE PRIMER TRANSFER-RNA
    BARAT, C
    LULLIEN, V
    SCHATZ, O
    KEITH, G
    NUGEYRE, MT
    GRUNINGERLEITCH, F
    BARRESINOUSSI, F
    LEGRICE, SFJ
    DARLIX, JL
    [J]. EMBO JOURNAL, 1989, 8 (11) : 3279 - 3285
  • [4] Berkowitz R, 1996, CURR TOP MICROBIOL, V214, P177
  • [5] Mutations in the N-terminal domain of human immunodeficiency virus type 1 nucleocapsid protein affect virion core structure and proviral DNA synthesis
    Berthoux, L
    Pechoux, C
    Ottmann, M
    Morel, G
    Darlix, JL
    [J]. JOURNAL OF VIROLOGY, 1997, 71 (09) : 6973 - 6981
  • [6] BOEKE JD, 1991, MOL CELLULAR BIOL YE, P193
  • [7] NATIVE RIBONUCLEOPROTEIN IS AN EFFICIENT TRANSCRIPTIONAL COMPLEX OF AVIAN-MYELOBLASTOSIS VIRUS
    CHEN, MJ
    GARON, CF
    PAPAS, TS
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1980, 77 (03): : 1296 - 1300
  • [8] COFFIN JM, 1985, RNA TUMOR VIRUSES, P261
  • [9] SPECIFIC BINDING OF HIV-1 NUCLEOCAPSID PROTEIN TO PSI-RNA IN-VITRO REQUIRES N-TERMINAL ZINC-FINGER AND FLANKING BASIC-AMINO-ACID RESIDUES
    DANNULL, J
    SUROVOY, A
    JUNG, G
    MOELLING, K
    [J]. EMBO JOURNAL, 1994, 13 (07) : 1525 - 1533
  • [10] CIS ELEMENTS AND TRANS-ACTING FACTORS INVOLVED IN THE RNA DIMERIZATION OF THE HUMAN-IMMUNODEFICIENCY-VIRUS HIV-1
    DARLIX, JL
    GABUS, C
    NUGEYRE, MT
    CLAVEL, F
    BARRESINOUSSI, F
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1990, 216 (03) : 689 - 699