Structural and functional properties of the HIV-1 RNA-tRNA(Lys)3 primer complex annealed by the nucleocapsid protein: comparison with the heat-annealed complex.
The conversion of the single-stranded RNA genome into double-stranded DNA by virus-coded reverse transcriptase (RT) is an essential step of the retrovirus life cycle. In human immunodeficiency virus type 1 (HIV-1), RT uses the cellular tRNA(Lys)3 to initiate the (-) strand DNA synthesis. Placement of the primer tRNA(Lys)3 involves binding of its 3'-terminal 18 nt to a complementary region of genomic RNA termed PBS. However, the PBS sequence is not the unique determinant of primer usage and additional contacts are important. This placement is believed to be achieved in vivo by the nucleocapsid domain of Gag or by the mature protein NCp. Up to now, structural information essentially arose from heat-annealed primer-template complexes (Isel et al., J Mol Biol, 1995, 247:236-250; Isel et al., EMBO J, 1999, 18:1038-1048). Here, we investigated the formation of the primer-template complex mediated by NCp and compared structural and functional properties of heat- and NCp-annealed complexes. We showed that both heat- and NCp-mediated procedures allow comparable high yields of annealing. Then, we investigated structural features of both kinds of complexes by enzymatic probing, and we compared their relative efficiency in (-) strong stop DNA synthesis. We did not find any significant differences between these complexes, suggesting that information derived from the heat-annealed complex can be transposed to the NCp-mediated complex and most likely to complexes formed in vivo.
Brulé
Fabienne
F
Unité Propre de Recherche 9002, Centre Nationale de la Recherche Scientifique, Institut de Biologie Moleculaire et Cellulaire, Strasbourg, France.
Marquet
Roland
R
Rong
Liwei
L
Wainberg
Mark A
MA
Roques
Bernard P
BP
Le Grice
Stuart F J
SF
Ehresmann
Bernard
B
Ehresmann
Chantal
C
D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication; D024202:Electrophoretic Mobility Shift Assay; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D015341:Nucleic Acid Probes; D019590:Nucleocapsid Proteins / Q000378:metabolism*; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012357:RNA, Transfer, Lys / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
D015341:Nucleic Acid Probes; D019590:Nucleocapsid Proteins; C034477:RNA primers; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D012313:RNA; D004247:DNA