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A proposal for a new HIV-1 DLS structural model.

Sakuragi J, Ode H, Sakuragi S, Shioda T, Sato H - Nucleic Acids Res. (2012)

Bottom Line: Interestingly, the study revealed a previously unnoticed stem formation between two distantly positioned regions.Based on this and other findings on functional base pairing in vivo, we propose new 3D models of the HIV-1 DLS which contain a unique pseudoknot-like conformation.Since this pseudoknot-like conformation appears to be thermodynamically stable, forms a foundational skeleton for the DLS and sterically restricts the spontaneous diversification of DLS conformations, its unique shape may contribute to the viral life cycle and potentially serve as a novel target for anti-HIV-1 therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Viral Infections, RIMD, Osaka Univ. 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. sakuragi@biken.osaka-u.ac.jp

ABSTRACT
The dimer initiation site/dimer linkage sequence (DIS/DLS) region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is suggested to play essential roles at various stages of the viral life cycle. Through a novel assay we had recently developed, we reported on the necessary and sufficient region for RNA dimerization in the HIV-1 virion. Using this system, we performed further detailed mapping of the functional base pairs necessary for HIV-1 DLS structure. Interestingly, the study revealed a previously unnoticed stem formation between two distantly positioned regions. Based on this and other findings on functional base pairing in vivo, we propose new 3D models of the HIV-1 DLS which contain a unique pseudoknot-like conformation. Since this pseudoknot-like conformation appears to be thermodynamically stable, forms a foundational skeleton for the DLS and sterically restricts the spontaneous diversification of DLS conformations, its unique shape may contribute to the viral life cycle and potentially serve as a novel target for anti-HIV-1 therapies.

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Related in: MedlinePlus

Confirmation of the stem formations within the DLS. (A) Schematic representations of the stems in HIV-1 DLS studied in this report. The specific positions examined are enclosed with rectangles and tagged with capitals. (B–E) The dimerization assay of the mutants. Upper tables describe the mutations introduced. Hyphens represent base deletions. Lower graphs represent dimerization ability of the mutants. Results are the average of at least three independent experiments with SEM.
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gks156-F4: Confirmation of the stem formations within the DLS. (A) Schematic representations of the stems in HIV-1 DLS studied in this report. The specific positions examined are enclosed with rectangles and tagged with capitals. (B–E) The dimerization assay of the mutants. Upper tables describe the mutations introduced. Hyphens represent base deletions. Lower graphs represent dimerization ability of the mutants. Results are the average of at least three independent experiments with SEM.

Mentions: Recently, in vivo HIV-1 RNA secondary structures have been described in several reports, based on the results of chemical or enzymatic probing, such as SHAPE analysis (10,33,34). In these presented structural models, there were no traces of the GACGC-GCGUC duplex. To get further information about the duplex by chemical probing, we have performed SHAPE analysis with ex virion RNA. The reactivity of the duplex mutants (NLAIS4m1, NLAIS4m1PK and NLAIC236G) along with the wild-type virus was calculated for each base in the duplex and adjacent U5-AUG duplex (Supplementary Figure S3A and B). Consistent with the previous SHAPE reports, the overall GACGC-GCGUC duplex reactivity was very low and not significantly affected by the mutations to destabilize (C236G and S4m1) and to restore the duplex (S4m1PK). Although it was very faint, there were some changes in reactivity, notably at GCGUC sequence exclusively in the destabilizing mutant. In addition, coinciding with the results from dimerization assay above (Figure 4B) and previous SHAPE assays, the 5′-end of DLS (U105-U107) was relatively reactive, suggesting the absence of base paring. At the same time, similar to the GACGC-GCGUC duplex, the overall U5-AUG duplex reactivity was not significantly affected by the mutations.Figure 4.


A proposal for a new HIV-1 DLS structural model.

Sakuragi J, Ode H, Sakuragi S, Shioda T, Sato H - Nucleic Acids Res. (2012)

Confirmation of the stem formations within the DLS. (A) Schematic representations of the stems in HIV-1 DLS studied in this report. The specific positions examined are enclosed with rectangles and tagged with capitals. (B–E) The dimerization assay of the mutants. Upper tables describe the mutations introduced. Hyphens represent base deletions. Lower graphs represent dimerization ability of the mutants. Results are the average of at least three independent experiments with SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367192&req=5

gks156-F4: Confirmation of the stem formations within the DLS. (A) Schematic representations of the stems in HIV-1 DLS studied in this report. The specific positions examined are enclosed with rectangles and tagged with capitals. (B–E) The dimerization assay of the mutants. Upper tables describe the mutations introduced. Hyphens represent base deletions. Lower graphs represent dimerization ability of the mutants. Results are the average of at least three independent experiments with SEM.
Mentions: Recently, in vivo HIV-1 RNA secondary structures have been described in several reports, based on the results of chemical or enzymatic probing, such as SHAPE analysis (10,33,34). In these presented structural models, there were no traces of the GACGC-GCGUC duplex. To get further information about the duplex by chemical probing, we have performed SHAPE analysis with ex virion RNA. The reactivity of the duplex mutants (NLAIS4m1, NLAIS4m1PK and NLAIC236G) along with the wild-type virus was calculated for each base in the duplex and adjacent U5-AUG duplex (Supplementary Figure S3A and B). Consistent with the previous SHAPE reports, the overall GACGC-GCGUC duplex reactivity was very low and not significantly affected by the mutations to destabilize (C236G and S4m1) and to restore the duplex (S4m1PK). Although it was very faint, there were some changes in reactivity, notably at GCGUC sequence exclusively in the destabilizing mutant. In addition, coinciding with the results from dimerization assay above (Figure 4B) and previous SHAPE assays, the 5′-end of DLS (U105-U107) was relatively reactive, suggesting the absence of base paring. At the same time, similar to the GACGC-GCGUC duplex, the overall U5-AUG duplex reactivity was not significantly affected by the mutations.Figure 4.

Bottom Line: Interestingly, the study revealed a previously unnoticed stem formation between two distantly positioned regions.Based on this and other findings on functional base pairing in vivo, we propose new 3D models of the HIV-1 DLS which contain a unique pseudoknot-like conformation.Since this pseudoknot-like conformation appears to be thermodynamically stable, forms a foundational skeleton for the DLS and sterically restricts the spontaneous diversification of DLS conformations, its unique shape may contribute to the viral life cycle and potentially serve as a novel target for anti-HIV-1 therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Viral Infections, RIMD, Osaka Univ. 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. sakuragi@biken.osaka-u.ac.jp

ABSTRACT
The dimer initiation site/dimer linkage sequence (DIS/DLS) region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is suggested to play essential roles at various stages of the viral life cycle. Through a novel assay we had recently developed, we reported on the necessary and sufficient region for RNA dimerization in the HIV-1 virion. Using this system, we performed further detailed mapping of the functional base pairs necessary for HIV-1 DLS structure. Interestingly, the study revealed a previously unnoticed stem formation between two distantly positioned regions. Based on this and other findings on functional base pairing in vivo, we propose new 3D models of the HIV-1 DLS which contain a unique pseudoknot-like conformation. Since this pseudoknot-like conformation appears to be thermodynamically stable, forms a foundational skeleton for the DLS and sterically restricts the spontaneous diversification of DLS conformations, its unique shape may contribute to the viral life cycle and potentially serve as a novel target for anti-HIV-1 therapies.

Show MeSH
Related in: MedlinePlus