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The Interaction between tRNA(Lys) 3 and the primer activation signal deciphered by NMR spectroscopy.

Sleiman D, Barraud P, Brachet F, Tisne C - PLoS ONE (2013)

Bottom Line: Despite the fact that the result of this rearrangement is thermodynamically more stable, there is a high-energy barrier that requires the chaperoning activity of the viral nucleocapsid protein.Our NMR study provides molecular evidence of the existence of this interaction and highlights the role of the nucleocapsid protein in promoting this additional RNA-RNA annealing.This study presents the first direct observation at a single base-pair resolution of the PAS/anti-PAS association, which has been proposed to be involved in the chronological regulation of the reverse transcription.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Cristallographie et RMN biologiques, CNRS, Université Paris Descartes, Paris Sorbonne Cité, Paris, France.

ABSTRACT
The initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) requires the opening of the three-dimensional structure of the primer tRNA(Lys) 3 for its annealing to the viral RNA at the primer binding site (PBS). Despite the fact that the result of this rearrangement is thermodynamically more stable, there is a high-energy barrier that requires the chaperoning activity of the viral nucleocapsid protein. In addition to the nucleotide complementarity to the PBS, several regions of tRNA(Lys) 3 have been described as interacting with the viral genomic RNA. Among these sequences, a sequence of the viral genome called PAS for "primer activation signal" was proposed to interact with the T-arm of tRNA(Lys) 3, this interaction stimulating the initiation of reverse transcription. In this report, we investigate the formation of this additional interaction with NMR spectroscopy, using a simple system composed of the primer tRNA(Lys) 3, the 18 nucleotides of the PBS, the PAS (8 nucleotides) encompassed or not in a hairpin structure, and the nucleocapsid protein. Our NMR study provides molecular evidence of the existence of this interaction and highlights the role of the nucleocapsid protein in promoting this additional RNA-RNA annealing. This study presents the first direct observation at a single base-pair resolution of the PAS/anti-PAS association, which has been proposed to be involved in the chronological regulation of the reverse transcription.

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Related in: MedlinePlus

Secondary structures of RNA used in this study.A) the recombinant tRNALys3 expressed in E. coli, B) the hairpin encompassing PAS sequence, the double-stranded PAS (PASds) with the numbering of nucleotides in the HXB2 isolate, C) the single-stranded PAS sequence (PASss) and D) the PBS. A cartoon symbolizing the secondary structure of these RNAs is drawn next their names.
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pone-0064700-g002: Secondary structures of RNA used in this study.A) the recombinant tRNALys3 expressed in E. coli, B) the hairpin encompassing PAS sequence, the double-stranded PAS (PASds) with the numbering of nucleotides in the HXB2 isolate, C) the single-stranded PAS sequence (PASss) and D) the PBS. A cartoon symbolizing the secondary structure of these RNAs is drawn next their names.

Mentions: A similar labelling strategy using the NMR signals of the imino protons of tRNALys3 as reporter signals has already been successfully employed to study the secondary structure of the HIV reverse transcription initiation complex and to reveal the different steps of annealing of tRNALys3 to the PBS in the presence or in the absence of the nucleocapsid protein [7], [38]. In the present study, tRNALys315N-labelling is also an appropriate tool to observe the annealing of the PAS and anti-PAS sequences (Figure 1C) as the largest number of sequential imino protons (carried by Gs and Us) belongs to tRNALys3, and can therefore be observed in 2D 1H-15N correlation spectra. The human tRNALys3 was thus produced as a recombinant tRNA in E. coli providing the incorporation of modified nucleotides by E. coli RNA modification enzymes as well as a uniform 15N labelling (see Materials and Methods). This recombinant tRNA (Figure 2A) bears the modified nucleotides crucial for the initiation of HIV-1 reverse transcription [21], [39]. In the case of the PAS activation, it was shown that reverse transcription primed by natural or synthetic tRNALys3 is similarly activated, indicating that the PAS/anti-PAS interaction is not dependent on modified nucleotides within tRNALys3[18].


The Interaction between tRNA(Lys) 3 and the primer activation signal deciphered by NMR spectroscopy.

Sleiman D, Barraud P, Brachet F, Tisne C - PLoS ONE (2013)

Secondary structures of RNA used in this study.A) the recombinant tRNALys3 expressed in E. coli, B) the hairpin encompassing PAS sequence, the double-stranded PAS (PASds) with the numbering of nucleotides in the HXB2 isolate, C) the single-stranded PAS sequence (PASss) and D) the PBS. A cartoon symbolizing the secondary structure of these RNAs is drawn next their names.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675109&req=5

pone-0064700-g002: Secondary structures of RNA used in this study.A) the recombinant tRNALys3 expressed in E. coli, B) the hairpin encompassing PAS sequence, the double-stranded PAS (PASds) with the numbering of nucleotides in the HXB2 isolate, C) the single-stranded PAS sequence (PASss) and D) the PBS. A cartoon symbolizing the secondary structure of these RNAs is drawn next their names.
Mentions: A similar labelling strategy using the NMR signals of the imino protons of tRNALys3 as reporter signals has already been successfully employed to study the secondary structure of the HIV reverse transcription initiation complex and to reveal the different steps of annealing of tRNALys3 to the PBS in the presence or in the absence of the nucleocapsid protein [7], [38]. In the present study, tRNALys315N-labelling is also an appropriate tool to observe the annealing of the PAS and anti-PAS sequences (Figure 1C) as the largest number of sequential imino protons (carried by Gs and Us) belongs to tRNALys3, and can therefore be observed in 2D 1H-15N correlation spectra. The human tRNALys3 was thus produced as a recombinant tRNA in E. coli providing the incorporation of modified nucleotides by E. coli RNA modification enzymes as well as a uniform 15N labelling (see Materials and Methods). This recombinant tRNA (Figure 2A) bears the modified nucleotides crucial for the initiation of HIV-1 reverse transcription [21], [39]. In the case of the PAS activation, it was shown that reverse transcription primed by natural or synthetic tRNALys3 is similarly activated, indicating that the PAS/anti-PAS interaction is not dependent on modified nucleotides within tRNALys3[18].

Bottom Line: Despite the fact that the result of this rearrangement is thermodynamically more stable, there is a high-energy barrier that requires the chaperoning activity of the viral nucleocapsid protein.Our NMR study provides molecular evidence of the existence of this interaction and highlights the role of the nucleocapsid protein in promoting this additional RNA-RNA annealing.This study presents the first direct observation at a single base-pair resolution of the PAS/anti-PAS association, which has been proposed to be involved in the chronological regulation of the reverse transcription.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Cristallographie et RMN biologiques, CNRS, Université Paris Descartes, Paris Sorbonne Cité, Paris, France.

ABSTRACT
The initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) requires the opening of the three-dimensional structure of the primer tRNA(Lys) 3 for its annealing to the viral RNA at the primer binding site (PBS). Despite the fact that the result of this rearrangement is thermodynamically more stable, there is a high-energy barrier that requires the chaperoning activity of the viral nucleocapsid protein. In addition to the nucleotide complementarity to the PBS, several regions of tRNA(Lys) 3 have been described as interacting with the viral genomic RNA. Among these sequences, a sequence of the viral genome called PAS for "primer activation signal" was proposed to interact with the T-arm of tRNA(Lys) 3, this interaction stimulating the initiation of reverse transcription. In this report, we investigate the formation of this additional interaction with NMR spectroscopy, using a simple system composed of the primer tRNA(Lys) 3, the 18 nucleotides of the PBS, the PAS (8 nucleotides) encompassed or not in a hairpin structure, and the nucleocapsid protein. Our NMR study provides molecular evidence of the existence of this interaction and highlights the role of the nucleocapsid protein in promoting this additional RNA-RNA annealing. This study presents the first direct observation at a single base-pair resolution of the PAS/anti-PAS association, which has been proposed to be involved in the chronological regulation of the reverse transcription.

Show MeSH
Related in: MedlinePlus