New insights into the formation of HIV-1 reverse transcription initiation complex

Abstract

HIV-1 reverse transcriptase uses the host tRNA 3 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 TΨC stems, and that, at physiological temperature, complete annealing is reached only in the presence of NC, probably when the zinc fingers contact the TΨC/D loops.In the present work, we have refined our model of the formation of the tRNA 3 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 TΨC 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 tRNA 3 Lys . Interestingly, the NC protein shows strong and specific binding to the D-arm of tRNA 3 Lys , which could explain the overall annealing mechanism.

Biophysical studies of the nucleic acid chaperone properties of the HIV-1 nucleocapsid protein

“…These data were reconciled in a refined model in which both the center of the cloverleaf and the 3'-end unpaired bases of the primer tRNA are used for PBS annealing. 113 However, it is still unclear whether the same viral RNA molecule can simultaneously invade the tRNA acceptor stem from both ends or whether some proteins initiate the annealing at the tRNA center while others "zip-in" from the 3'-end, in a statistical manner.…”

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

“…The TAR sequence used was that of the NL4-3 isolate. The NC(1-55) sequence (NL4-3 isolate) was overexpressed and the product was purified, as previously described (Barraud et al 2007).…”

The HIV-1 Nucleocapsid Protein Recruits Negatively Charged Lipids To Ensure Its Optimal Binding to Lipid Membranes

“…Mature NC results from the protease-mediated cleavage of the Gag polyprotein precursor and plays key roles in HIV-1 replication (2)(3)(4)(5). During the early steps, NC acts as a cofactor of the reverse transcriptase (RT), to promote the initiation of reverse transcription (6)(7)(8), as well as the two obligatory strand transfers (9,10). Moreover, NC also reduces RT pauses at the initiation step (11)(12)(13), increases the overall RT processivity (14,15), promotes synthesis through pause sites (16)(17)(18)(19), helps to remove the RNA fragments resulting from the RT RNase H activity (20), and may contribute to the protection of the viral DNA (vDNA) during its nuclear import (21).…”