New insights into the formation of HIV-1 reverse transcription initiation complex.
HIV-1 reverse transcriptase uses the host tRNA3(Lys) as a primer for the synthesis of the minus DNA strand. The first event in viral replication is thus the annealing of tRNA to the primer binding site (PBS) in the 5' UTR of the viral RNA. This event requires a major RNA rearrangement which is chaperoned by the viral NC protein. The binding of NC to nucleic acids is essentially non-specific, however, NC is known to bind selectively to hairpins located in the 5' region of the viral RNA. In a previous study, using an NMR approach in which the reaction is slowed down by controlling temperature, we were able to follow details in this RNA unfolding/refolding process and to uncover an intermediate state. We showed that annealing initiates at the junction between the acceptor and the TPsiC stems, and that, at physiological temperature, complete annealing is reached only in the presence of NC, probably when the zinc fingers contact the TPsiC/D loops. In the present work, we have refined our model of the formation of the tRNA3(Lys)/PBS duplex. First, we show that annealing can initiate both from the single-stranded CCA 3'-end bases of the acceptor stem and from the bases in the TPsiC stem. Secondly, by NMR and fluorescence spectroscopy, we have studied the complex between the NC protein and RNA hairpins that mimic the D and T arms of the tRNA3(Lys). Interestingly, the NC protein shows strong and specific binding to the D arm of tRNA3(Lys), which could explain the overall annealing mechanism.
Barraud
Pierre
P
Laboratoire de Cristallographie et RMN biologiques, Université Paris Descartes, CNRS, UMR 8015, 4 avenue de l'Observatoire, 75006 Paris, France.
Gaudin
Cyril
C
Dardel
Frédéric
F
Tisné
Carine
C
D001483:Base Sequence; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000378:metabolism; D012357:RNA, Transfer, Lys / Q000737:chemistry / Q000235:genetics; D048348:Reverse Transcription / Q000235:genetics*
D019590:Nucleocapsid Proteins; D012357:RNA, Transfer, Lys