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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

Thermodynamic stability of the interaction involving the PBS (in blue) or the PAS (in red) sequences.ΔG values calculated with UNAFold [57] are indicated in kcal/mol and the number of base pairs are indicated in parenthesis. The melting temperature Tmc was calculated by UNAFold and the melting temperature Tm was measured by UV spectroscopy (Figure S5). See also Figure S1 for details on the different sequence and secondary structures used to model the intra-molecular interaction within the MAL and HXB2 isolates.
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pone-0064700-g005: Thermodynamic stability of the interaction involving the PBS (in blue) or the PAS (in red) sequences.ΔG values calculated with UNAFold [57] are indicated in kcal/mol and the number of base pairs are indicated in parenthesis. The melting temperature Tmc was calculated by UNAFold and the melting temperature Tm was measured by UV spectroscopy (Figure S5). See also Figure S1 for details on the different sequence and secondary structures used to model the intra-molecular interaction within the MAL and HXB2 isolates.

Mentions: From a thermodynamic point of view, the annealing of tRNALys3 to the PBS is strongly favoured (Figure 5, ΔG  = −36.2 kcal/mol and Tmc  = 92°C). In addition, the annealing of tRNALys3 to the PBS will partially open the tRNA structure and thus increase the accessibility to the anti-PAS sequence. Therefore, in order to investigate whether the PBS could influence the annealing of the PAS sequence with tRNALys3, we decided to conduct the heat-annealed procedure in the presence of the PBS. Figure 6 shows the NMR footprint experiment resulting from this procedure. The assignment of tRNALys3/PBS imino groups was previously published [7]. The imino groups of tRNALys3 (black spectrum – Figure 6) have disappeared to give rise to imino groups of tRNALys3 involved in base-pairing with the PBS (red spectrum – Figure 6). A NOESY experiment (data not shown) substantiates that the PASds is still free as its imino groups are still observable at the chemical shifts of the imino groups of the free PASds like in Figure 4. In conclusion, when we mixed 15N-tRNALys3 to the PBS and to the PASds at the same time, the heat-annealed complex is composed of the PBS bound to tRNALys3 and the PAS remains free in solution (Figure 6). This complex is stable and does not change with time.


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)

Thermodynamic stability of the interaction involving the PBS (in blue) or the PAS (in red) sequences.ΔG values calculated with UNAFold [57] are indicated in kcal/mol and the number of base pairs are indicated in parenthesis. The melting temperature Tmc was calculated by UNAFold and the melting temperature Tm was measured by UV spectroscopy (Figure S5). See also Figure S1 for details on the different sequence and secondary structures used to model the intra-molecular interaction within the MAL and HXB2 isolates.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0064700-g005: Thermodynamic stability of the interaction involving the PBS (in blue) or the PAS (in red) sequences.ΔG values calculated with UNAFold [57] are indicated in kcal/mol and the number of base pairs are indicated in parenthesis. The melting temperature Tmc was calculated by UNAFold and the melting temperature Tm was measured by UV spectroscopy (Figure S5). See also Figure S1 for details on the different sequence and secondary structures used to model the intra-molecular interaction within the MAL and HXB2 isolates.
Mentions: From a thermodynamic point of view, the annealing of tRNALys3 to the PBS is strongly favoured (Figure 5, ΔG  = −36.2 kcal/mol and Tmc  = 92°C). In addition, the annealing of tRNALys3 to the PBS will partially open the tRNA structure and thus increase the accessibility to the anti-PAS sequence. Therefore, in order to investigate whether the PBS could influence the annealing of the PAS sequence with tRNALys3, we decided to conduct the heat-annealed procedure in the presence of the PBS. Figure 6 shows the NMR footprint experiment resulting from this procedure. The assignment of tRNALys3/PBS imino groups was previously published [7]. The imino groups of tRNALys3 (black spectrum – Figure 6) have disappeared to give rise to imino groups of tRNALys3 involved in base-pairing with the PBS (red spectrum – Figure 6). A NOESY experiment (data not shown) substantiates that the PASds is still free as its imino groups are still observable at the chemical shifts of the imino groups of the free PASds like in Figure 4. In conclusion, when we mixed 15N-tRNALys3 to the PBS and to the PASds at the same time, the heat-annealed complex is composed of the PBS bound to tRNALys3 and the PAS remains free in solution (Figure 6). This complex is stable and does not change with time.

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