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. 2016 Nov;22(11):1689-1698.
doi: 10.1261/rna.056804.116. Epub 2016 Sep 9.

Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site

Aaron Coey  1   2 , Kevin Larsen  1   2 , Joseph D Puglisi  1 , Elisabetta Viani Puglisi  1
Affiliations

Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site

Aaron Coey et al. RNA. 2016 Nov.

Abstract

Reverse transcription is a key process in the early steps of HIV infection. This process initiates within a specific complex formed by the 5' UTR of the HIV genomic RNA (vRNA) and a host primer tRNALys3 Using nuclear magnetic resonance (NMR) spectroscopy and single-molecule fluorescence spectroscopy, we detect two distinct conformers adopted by the tRNA/vRNA initiation complex. We directly show that an interaction between the conserved 8-nucleotide viral RNA primer activation signal (PAS) and the primer tRNA occurs in one of these conformers. This intermolecular PAS interaction likely induces strain on a vRNA intramolecular helix, which must be broken for reverse transcription to initiate. We propose a mechanism by which this vRNA/tRNA conformer relieves the kinetic block formed by the vRNA intramolecular helix to initiate reverse transcription.

Keywords: HIV RNA structure; NMR spectroscopy; reverse transcription; single-molecule FRET.

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Figures

FIGURE 1.
FIGURE 1.
(A) The primer tRNALys3 anneals to the vRNA at the PBS site to form an 18-bp helix from which reverse transcription initiates. This site is found within the highly structured 5′ UTR. The proximal locations of the conserved vRNA PBS and PAS sequences are highlighted in red and blue, respectively. (B) The sequences of the 77-nt-vRNA construct and tRNALys3 primer contain the complementary PBS/anti-PBS and PAS/anti-PAS sequences highlighted in red and blue, respectively.
FIGURE 2.
FIGURE 2.
(A) The homonuclear spectrum of the 69-nt-vRNA/tRNA complex (red) overlaid on the 77-nt-vRNA/tRNA spectrum (black) shows that the 77-nt-vRNA/tRNA complex adopts a fold similar to the 69-nt-vRNA/tRNA spectrum. Several new NOEs within the 77-nt-vRNA/tRNA spectrum do not overlap with the 69-nt-vRNA/tRNA spectrum. (B) The 15N-1H TROSY spectrum of the 69-nt-vRNA/tRNA complex containing 15N-labeled tRNA (red) overlaid on the 77-nt-vRNA/tRNA 15N-1H TROSY spectrum containing 15N-labeled tRNA (black) shows that the tRNA adopts a similar fold when in complex with either 69-nt-vRNA or 77-nt-VRNA. (C) Secondary structure of the 77-nt-vRNA/tRNA complex showing a similar fold to the 69-nt-vRNA/tRNA complex.
FIGURE 3.
FIGURE 3.
(A) The 5′ side of the PAS oligo was constructed from the vRNA PAS sequence and the 3′ side from the tRNA anti-PAS sequence. The stem is capped by a UUCG tetraloop on one end and a GC base pair on the other. (B) Assigning the homonuclear NOESY spectrum of the PAS oligo shows that it folds into a single stem–loop structure. (C) Overlaying the PAS oligo NOESY spectrum (red) onto the 77-nt-vRNA/tRNA NOESY spectrum (black) shows that the 77-nt-vRNA/tRNA complex contains a PAS/anti-PAS secondary structural interaction. (D) NOESY data show that the vRNA PAS sequence displaces the tRNA 5′ end to base pair with the tRNA anti-PAS sequence.
FIGURE 4.
FIGURE 4.
Placing donor and acceptor dyes at the 5′ and 3′ ends of the 77-nt-vRNA, respectively, allows the direct monitoring of PAS/anti-PAS interactions. A low FRET signal indicates that the PAS/anti-PAS interaction is not present, whereas a high FRET signal indicates that the PAS/anti-PAS sequences have annealed.
FIGURE 5.
FIGURE 5.
(A) Two distinct FRET states were observed for the wild-type 77-nt-vRNA/tRNA FRET complex. The low FRET state (above) at 0.4 FRET efficiency was attributed to the intramolecular anti-PAS conformation, and the state (below) at 1.0 FRET efficiency was attributed to the intermolecular PAS conformation. (B) Approximately one-third of the wild-type complexes is in the low FRET intramolecular anti-PAS conformation while two-thirds are in the high FRET intermolecular PAS conformation (n = 167). The tRNA-ΔA50 mutation completely ablates the high FRET state (n = 172), and the tRNA-Δ1-5 mutation shifts the majority of the complexes to the high FRET state (n = 233).
FIGURE 6.
FIGURE 6.
The 15N-1H TROSY spectrum of 15N-labeled 77-nt-vRNA in complex with unlabeled tRNA-ΔA50 (red) overlaid onto the 15N-1H TROSY spectrum of 15N-labeled 69-nt-vRNA in complex with unlabeled wild-type tRNA (black) shows that the formation of the intermolecular PAS interaction maintains the vRNA intramolecular helix. Resonances belonging to imino protons in the vRNA intramolecular helix are labeled.
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