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. 2011 Jul 29;410(5):863-74.
doi: 10.1016/j.jmb.2011.04.024.

Secondary structure of the HIV reverse transcription initiation complex by NMR

Elisabetta Viani Puglisi  1 , Joseph D Puglisi
Affiliations

Secondary structure of the HIV reverse transcription initiation complex by NMR

Elisabetta Viani Puglisi et al. J Mol Biol. .

Abstract

Initiation of reverse transcription of genomic RNA is a key early step in replication of the human immunodeficiency virus (HIV) upon infection of a host cell. Viral reverse transcriptase initiates from a specific RNA-RNA complex formed between a host transfer RNA (tRNA(Lys)(3)) and a region at the 5' end of genomic RNA; the 3' end of the tRNA acts as a primer for reverse transcription of genomic RNA. We report here the secondary structure of the HIV genomic RNA-human tRNA(Lys)(3) initiation complex using heteronuclear nuclear magnetic resonance methods. We show that both RNAs undergo large-scale conformational changes upon complex formation. Formation of the 18-bp primer helix with the 3' end of tRNA(Lys)(3) drives large conformational rearrangements of the tRNA at the 5' end while maintaining the anticodon loop for potential loop-loop interactions. HIV RNA forms an intramolecular helix adjacent to the intermolecular primer helix. This helix, which must be broken by reverse transcription, likely acts as a kinetic block to reverse transcription.

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Figures

Figure 1
Figure 1
(A.) Schematic of the reverse transcription initiation complex within the HIV genome. The RNA genome of HIV RNA has the standard structure for retroviral genomes, with long terminal repeat regions (U5, U3, R), and the primer binding site (PBS) near the 5'end. Host tRNALys3 interacts with the PBS through an 18 base-pair interaction, providing the 3'-OH group to initiate reverse transcription by viral reverse transcriptase (RT). (B.) RNA oligonucleotides corresponding to HIV-1 genomic RNAs used in the current study (left) a 99 nt and (right) 69 nt RNA with numbering according to the Mal isolate. Additional nucleotides added at the 5'end for transcription with T7 RNA polymerase are indicated as outlines. The primer binding site region of 18bp of complementarity with tRNA is highlighted in red, whereas the A-rich loop is in yellow. (c) Secondary structure of human tRNALys3 with modified nucleotides; Coloring as in (b).
Figure 2
Figure 2. Chemical probing experiments on HIV initiation complexes with 69nt model HIV1 genomic RNA
Accessibility of adenosine N1 and cytosine N3 positions to reaction with dimethyl sulfate (DMS) was detected in the presence or absence of 1:1 stoichiometry of either pure bovine native tRNALys3 (nat) or T7 RNA polymerase transcript (T7). Reactivity with DMS was detected using primer extension with reverse transcription using a DNA primer in the absence (−) and presence (+) of dimethyl sulfate reaction. Reactions were performed at room temperature as described.
Figure 3
Figure 3. Imino 1H NMR spectra of folded human tRNA Lys3 transcript (bottom) compared to that of 1:1 complex of human tRNALys3 with 69nt HIV1 genomic RNA (top)
Spectra were acquired at 800 MHz in 10mM MgCl2, 100mM NaCl, 10mM Na phosphate, pH 6.5 at 25°C. Assignments of resonances in folded tRNALys3 were reported previously.
Figure 4
Figure 4. Changes in tRNALys3 conformation upon initiation complex formation
(A.) 15N-1H TROSY spectrum of uniformly 13C, 15N labeled human tRNALys3 either alone (red) or in 1:1 complex with unlabeled HIV-1 69nt RNA (black). The comparison of the two spectra highlights the large changes in tRNA structure that occurs upon complex formation. (B.) Secondary structure of human tRNALys3; major perturbations (pink circles) of the folded tRNA spectrum upon complex formation with HIV RNA occur in the acceptor, T and D-stems, whereas resonances for the anticodon stem are relatively unperturbed. The spectra were acquired at 800 MHz in 10mM MgCl2 50mM NaCl, 10mM Na phosphate, pH 6.5 at 25°C using a T1-relaxation optimized TROSY pulse sequence as described in the text for improved signal-to-noise.
Figure 5
Figure 5. 1H-15N TROSY experiments for the HIV initiation complex at 1:1 tRNALys3-HIV RNA stoichiometry
(A.) Spectrum for initiation complex formed with uniformly labeled 13C, 15N-tRNALys3 and unlabeled HIV-1 69nt RNA. (B.) Spectrum for initiation complex formed with uniformly labeled 13C, 15N-HIV-1 69nt RNA and unlabeled tRNALys3. Spectra were acquired at 800 MHz 1H frequency T1-relaxation optimized TROSY at 25°C. Spectral assignments of the imino resonances as discussed in the text are indicated.
Figure 6
Figure 6. NOESY spectrum of unlabeled 1:1 complex of human tRNALys3 and HIV1 69nt RNA
The region of imino 1H-1H NOES is shown; data were acquired at 800 MHz with 100ms mixing time at 25°C. NOE connectivities are observed that allow assignment of 16 of the 18 base pairs formed between the tRNA and HIV RNA.
Figure 7
Figure 7. H-NN COSY experiments showing through-space 15N-15N scalarcouplings across base-pairing hydrogen bonds
(A.) H-NN COSY spectrum of the initiation complex formed with uniformly labeled 13C, 15N-tRNALys3 and unlabeled HIV-1 69nt RNA. (B.) H-NN COSY spectrum for initiation complex formed with uniformly labeled 13C, 15N-HIV-1 69nt RNA and unlabeled tRNALys3. Both experiments only show correlations for intramolecular base pairing for the 15N-labeled component of the complex. Data were acquired at 25°C at 800 MHz using a T1-relaxation optimized (BEST-HNN COSY) version that uses band selective pulses centered on the imino proton resonances as described in the materials and methods. (C). Secondary Structure of the HIV initiation complex determined here by NMR spectroscopy. HIV 1 genomic RNA (Mal-1 isolate) is in black, tRNALys3 in red. The key regions of the structure, including 18 bp PBS helix, HIV genomic RNA intramolecular helix, and the conservation of the tRNA anticodon stem-loop are highlighted. Potential U-rich anticodon loop interaction with A-rich sequence in the HIV RNA is shaded. Arrows indicate pausing sites observed in kinetic investigations of the HIV-1 initiation complex with reverse transcriptase. These strong pauses are at positions +3, +6 and +16.
Figure 7
Figure 7. H-NN COSY experiments showing through-space 15N-15N scalarcouplings across base-pairing hydrogen bonds
(A.) H-NN COSY spectrum of the initiation complex formed with uniformly labeled 13C, 15N-tRNALys3 and unlabeled HIV-1 69nt RNA. (B.) H-NN COSY spectrum for initiation complex formed with uniformly labeled 13C, 15N-HIV-1 69nt RNA and unlabeled tRNALys3. Both experiments only show correlations for intramolecular base pairing for the 15N-labeled component of the complex. Data were acquired at 25°C at 800 MHz using a T1-relaxation optimized (BEST-HNN COSY) version that uses band selective pulses centered on the imino proton resonances as described in the materials and methods. (C). Secondary Structure of the HIV initiation complex determined here by NMR spectroscopy. HIV 1 genomic RNA (Mal-1 isolate) is in black, tRNALys3 in red. The key regions of the structure, including 18 bp PBS helix, HIV genomic RNA intramolecular helix, and the conservation of the tRNA anticodon stem-loop are highlighted. Potential U-rich anticodon loop interaction with A-rich sequence in the HIV RNA is shaded. Arrows indicate pausing sites observed in kinetic investigations of the HIV-1 initiation complex with reverse transcriptase. These strong pauses are at positions +3, +6 and +16.
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