New insights into the formation of HIV-1 reverse transcription initiation complex
Introduction
In retroviruses, initiation of reverse transcription is primed by a cellular tRNA that is encapsidated in viral particles. tRNA3Lys is the natural primer of all immunodeficiency viruses, including the type 1 human immunodeficiency virus (HIV-1) [1], [2], [3]. The HIV-1 nucleocapsid protein (NC) is a short, basic, nucleic acid binding protein with two zinc finger domains, each containing the invariant CCHC metal-ion binding motif. The mature protein (55 amino acid residues) is produced by proteolytic cleavage of the Gag precursor and is found in the interior of the virus particle, where it is tightly associated with genomic RNA. NC or the NC domain in Gag is involved in multiple functions during the virus replication cycle, including genomic RNA packaging, primer placement on viral RNA, reverse transcription, and integration. Many of these functions rely on the nucleic acid chaperone activity of NC, i.e. its ability to catalyse nucleic acid conformational rearrangements that lead to the most thermodynamically stable structure (for recent reviews [4], [5], [6]).
In a previous study [7], we were able to observe the progressive formation of the HIV-1 reverse transcription initiation complex using heteronuclear NMR of RNA–RNA–protein complexes under controlled temperature conditions. In particular, we have identified a nucleation site, at the end of the acceptor stem, where the viral RNA starts invading the tRNA3Lys structure. In addition, we were also able to characterize the different roles of the nucleocapsid protein during the formation of the initiation complex. In our model, the viral NC protein plays a key role by “unlocking” stable 3D structure of the primer tRNA through specific interaction with the D/TΨC loops. Therefore, at the end of this study, the question of the involvement of the 3′-end unpaired CCA bases of tRNA3Lys primer in the annealing process was still open. In addition, the mechanism of the specific 3D structure opening was still not clearly understood.
In the present report, we show that the annealing can proceed from two starting points in the tRNA3Lys primer, one at the beginning of the acceptor stem and the second at the end of the T stem. Both sites can be used by the viral RNA to begin its annealing with the 18 complementary bases of tRNA3Lys acceptor and T stems. The presence of NC protein is not necessary at this step, at least in vitro, but is essential to open the 3D structure of tRNA. The melting of the interaction at the level of the D/TΨC loops that locks the structure of the tRNA is possible through a specific interaction between the D loop and the NC protein.
Section snippets
Samples
The PBS (18 nucleotides) was 15N-labelled on its guanine nucleotides by in vitro transcription from an oligonucleotide template containing a 2′-O-methyl-G in position 2 (DNA template: 5′-G(2′OmeG)TCCCTGTTCGGGCGCCACTATAGTGAGTCGTATT-3′) [8]. Labelled nucleotides were purchased from Spectra Gases Inc. The 20 nucleotides RNA obtained was then purified by electrophoresis. After electroelution and ethanol precipitation, the RNA was resuspended, micro-dialysed against a first buffer (10 mM sodium
Where are the nucleation sites for tRNA3Lys/PBS duplex formation?
In a previous study [7], we showed that the melting process starts at the junction between the acceptor and T stems, at the level of weak AU and GU base pairs which can be easily disrupted and hence allow subsequent invasion of the tRNA3Lys structure by the viral RNA. However, our experimental approach, which relied on the NMR observation of imino protons, did not allow observation of PBS binding to the unpaired 3′-end of the tRNA, since the imino protons of the first three base pairs are
Conclusion
In this paper, the use of selective labelling of the primer binding site allowed us to characterize the nucleation points of the annealing process between the PBS and the tRNA3Lys. Moreover, we used small hairpins to mimic the D and T arms of tRNA3Lys to determine the specific role of NC in the formation of tRNA3Lys/PBS duplex recognized by the HIV reverse transcriptase. Studying separate hairpins cannot obviously mimic the entire tRNA, in particular at the level of the TΨC/D loops interaction.
Acknowledgements
P. Barraud is supported by a studentship from Ministère de la Recherche. This work was supported by the French AIDS national Agency (ANRS) and ‘Ensemble contre le SIDA’ (Sidaction).
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Secondary structure of the HIV reverse transcription initiation complex by NMR
2011, Journal of Molecular BiologyCitation Excerpt :The first step in viral replication after entry into an infected cell is reverse transcription, which is catalyzed by a viral enzyme, reverse transcriptase (RT).1 RT must initiate from a specific RNA assembly and then navigate the complex secondary structure of genomic RNA while copying RNA to DNA.2 RT is a major target of therapeutic intervention in treatment of acquired immune deficiency syndrome.