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Inhibition of HIV-1 gene expression by Sam68 Delta C: multiple targets but a common mechanism?

Cochrane A - Retrovirology (2009)

Bottom Line: Two recent publications have explored the mechanisms by which a mutant of the host protein Sam68 blocks HIV-1 structural protein synthesis and expands its activity to encompass Nef.Although the two studies propose different mechanisms for the responses observed, it is possible that a common activity is responsible.Understanding how this Sam68 mutant discriminates among the multiple viral mRNAs promises to reveal unique properties of HIV-1 RNA metabolism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. alan.cochrane@utoronto.ca

ABSTRACT
Two recent publications have explored the mechanisms by which a mutant of the host protein Sam68 blocks HIV-1 structural protein synthesis and expands its activity to encompass Nef. Although the two studies propose different mechanisms for the responses observed, it is possible that a common activity is responsible. Understanding how this Sam68 mutant discriminates among the multiple viral mRNAs promises to reveal unique properties of HIV-1 RNA metabolism.

Show MeSH
Understanding regulation of HIV-1 gene expression by Sam68ΔC. (A) Following transcription, HIV-1 RNA undergoes alternative splicing to generate over 40 mRNAs that correspond to unspliced (encoding Gag and Gagpol), singly spliced (to produce Vif, Vpr, Vpu and Env) or multiply spliced (for generating Tat, Rev and Nef) mRNAs. Unspliced and singly spliced viral RNAs are exported to the cytoplasm via exportin-1, which is mediated by Rev, while the multiply spliced RNAs exit using Nxf1. Once within the cytoplasm, Sam68ΔC interacts with the unspliced, singly spliced and nef mRNAs to block their translation by preventing the binding of PABP1 (shown as a small blue circle). In contrast, PABP1 binds to tat and rev mRNAs, and translation is unaffected. (B) A model for the discrimination between tat, rev and nef mRNAs. The process of splicing used to generate the mRNAs encoding Tat, Rev and Nef results in slight variations in 5' sequence, but all the mRNAs encompass the nef reading frame (individual reading frames are illustrated by block arrows). However, translation of the individual reading frames could result in variations in the composition/structure of the mRNA within the common sequence (as represented by the coloured ovals). Such differences in composition/structure of the viral mRNP could serve as means by which Sam68ΔC selectively regulates their expression.
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Figure 1: Understanding regulation of HIV-1 gene expression by Sam68ΔC. (A) Following transcription, HIV-1 RNA undergoes alternative splicing to generate over 40 mRNAs that correspond to unspliced (encoding Gag and Gagpol), singly spliced (to produce Vif, Vpr, Vpu and Env) or multiply spliced (for generating Tat, Rev and Nef) mRNAs. Unspliced and singly spliced viral RNAs are exported to the cytoplasm via exportin-1, which is mediated by Rev, while the multiply spliced RNAs exit using Nxf1. Once within the cytoplasm, Sam68ΔC interacts with the unspliced, singly spliced and nef mRNAs to block their translation by preventing the binding of PABP1 (shown as a small blue circle). In contrast, PABP1 binds to tat and rev mRNAs, and translation is unaffected. (B) A model for the discrimination between tat, rev and nef mRNAs. The process of splicing used to generate the mRNAs encoding Tat, Rev and Nef results in slight variations in 5' sequence, but all the mRNAs encompass the nef reading frame (individual reading frames are illustrated by block arrows). However, translation of the individual reading frames could result in variations in the composition/structure of the mRNA within the common sequence (as represented by the coloured ovals). Such differences in composition/structure of the viral mRNP could serve as means by which Sam68ΔC selectively regulates their expression.

Mentions: Initial experiments [7] identified Sam68ΔC as a dominant inhibitor of HIV-1 replication. While subsequent work determined that inhibition was dependent upon the cytoplasmic localization of Sam68ΔC and associated with formation of cytoplasmic granules around the outside of the nuclear envelope [8], the underlying mechanism remained unclear. However, the recent work of Marsh et al. [6] provided some detail as to the mechanism. Using various expression vectors, they showed that Sam68ΔC selectively inhibited mRNA expressing Gag exported via the exportin-1 pathway, with little to no effect on the same Gag coding sequence delivered to the cytoplasm by Nxf1. Co-expression of Sam68ΔC and an unspliced Env expressor resulted in translation inhibition of the latter and disruption of the cytoplasmic bundles failed to restore expression of the encoded protein. Rather, despite a normal polyA tail, inhibition by Sam68ΔC was attributed to a block in translation of the affected RNA due to reduced binding of PABP-1 (Fig. 1A). The ability of Sam68ΔC to selectively affect only those RNAs exported in a Rev- and exportin-1-dependent fashion suggested that it recognizes some features unique to the mRNPs exported by this pathway. In parallel work by Henao-Mejia et al. [5] and consistent with Marsh et al., it was shown that constructs functionally similar to Sam68ΔC had the capacity to repress Rev-dependent protein expression. Surprisingly, inhibition of Rev-independent Nef synthesis was also observed with little or no alteration in Tat or Rev levels. Given that these three proteins are expressed from multiply spliced HIV-1 RNAs that all use the Nxf1 export pathway (Fig. 1A), selective repression of Nef expression may require a different mechanism than that outlined by Marsh et al. Inhibition of Nef expression was reported to be associated with the accumulation of nef mRNA in cytoplasmic granules that co-stained with markers of stress granules (SGs); these observations led Henao-Mejia et al. to suggest that reduced Nef synthesis was due to sequestration in these bodies. At present, it is unclear whether the granules characterized by Henao-Mejia et al. are similar or distinct from those formed by Sam68ΔC and incompletely spliced HIV-1 RNAs and whether Sam68ΔC inhibition of Nef synthesis is dependent upon their integrity. The two studies suggest that, while the route different RNAs take to repressive sites can differ (the Exportin1 pathway for Rev-dependent RNAs versus the Nxf1 pathway for nef mRNA), a similar mechanism may underlie repression of HIV-1 structural protein and nef mRNAs by Sam68ΔC. However, whether the mechanism is simple sequestration in SGs or something more complex remains to be determined. This is based on the observation of Marsh et al. that RRE-containing RNAs are still repressed upon dispersal of Sam68ΔC granules, although dispersion into functional "nano" granules cannot be dismissed and should be investigated. In addition, ongoing studies (Marsh and Cochrane, unpublished) showing that Sam68ΔC-induced granules contain mRNAs whose expression is not repressed suggest that sequestration to such granules alone is insufficient to explain translational repression. Consequently, additional experiments are needed to assess whether common or distinct mechanisms underlie repression of HIV-1 structural protein and nef mRNAs by Sam68ΔC.


Inhibition of HIV-1 gene expression by Sam68 Delta C: multiple targets but a common mechanism?

Cochrane A - Retrovirology (2009)

Understanding regulation of HIV-1 gene expression by Sam68ΔC. (A) Following transcription, HIV-1 RNA undergoes alternative splicing to generate over 40 mRNAs that correspond to unspliced (encoding Gag and Gagpol), singly spliced (to produce Vif, Vpr, Vpu and Env) or multiply spliced (for generating Tat, Rev and Nef) mRNAs. Unspliced and singly spliced viral RNAs are exported to the cytoplasm via exportin-1, which is mediated by Rev, while the multiply spliced RNAs exit using Nxf1. Once within the cytoplasm, Sam68ΔC interacts with the unspliced, singly spliced and nef mRNAs to block their translation by preventing the binding of PABP1 (shown as a small blue circle). In contrast, PABP1 binds to tat and rev mRNAs, and translation is unaffected. (B) A model for the discrimination between tat, rev and nef mRNAs. The process of splicing used to generate the mRNAs encoding Tat, Rev and Nef results in slight variations in 5' sequence, but all the mRNAs encompass the nef reading frame (individual reading frames are illustrated by block arrows). However, translation of the individual reading frames could result in variations in the composition/structure of the mRNA within the common sequence (as represented by the coloured ovals). Such differences in composition/structure of the viral mRNP could serve as means by which Sam68ΔC selectively regulates their expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651844&req=5

Figure 1: Understanding regulation of HIV-1 gene expression by Sam68ΔC. (A) Following transcription, HIV-1 RNA undergoes alternative splicing to generate over 40 mRNAs that correspond to unspliced (encoding Gag and Gagpol), singly spliced (to produce Vif, Vpr, Vpu and Env) or multiply spliced (for generating Tat, Rev and Nef) mRNAs. Unspliced and singly spliced viral RNAs are exported to the cytoplasm via exportin-1, which is mediated by Rev, while the multiply spliced RNAs exit using Nxf1. Once within the cytoplasm, Sam68ΔC interacts with the unspliced, singly spliced and nef mRNAs to block their translation by preventing the binding of PABP1 (shown as a small blue circle). In contrast, PABP1 binds to tat and rev mRNAs, and translation is unaffected. (B) A model for the discrimination between tat, rev and nef mRNAs. The process of splicing used to generate the mRNAs encoding Tat, Rev and Nef results in slight variations in 5' sequence, but all the mRNAs encompass the nef reading frame (individual reading frames are illustrated by block arrows). However, translation of the individual reading frames could result in variations in the composition/structure of the mRNA within the common sequence (as represented by the coloured ovals). Such differences in composition/structure of the viral mRNP could serve as means by which Sam68ΔC selectively regulates their expression.
Mentions: Initial experiments [7] identified Sam68ΔC as a dominant inhibitor of HIV-1 replication. While subsequent work determined that inhibition was dependent upon the cytoplasmic localization of Sam68ΔC and associated with formation of cytoplasmic granules around the outside of the nuclear envelope [8], the underlying mechanism remained unclear. However, the recent work of Marsh et al. [6] provided some detail as to the mechanism. Using various expression vectors, they showed that Sam68ΔC selectively inhibited mRNA expressing Gag exported via the exportin-1 pathway, with little to no effect on the same Gag coding sequence delivered to the cytoplasm by Nxf1. Co-expression of Sam68ΔC and an unspliced Env expressor resulted in translation inhibition of the latter and disruption of the cytoplasmic bundles failed to restore expression of the encoded protein. Rather, despite a normal polyA tail, inhibition by Sam68ΔC was attributed to a block in translation of the affected RNA due to reduced binding of PABP-1 (Fig. 1A). The ability of Sam68ΔC to selectively affect only those RNAs exported in a Rev- and exportin-1-dependent fashion suggested that it recognizes some features unique to the mRNPs exported by this pathway. In parallel work by Henao-Mejia et al. [5] and consistent with Marsh et al., it was shown that constructs functionally similar to Sam68ΔC had the capacity to repress Rev-dependent protein expression. Surprisingly, inhibition of Rev-independent Nef synthesis was also observed with little or no alteration in Tat or Rev levels. Given that these three proteins are expressed from multiply spliced HIV-1 RNAs that all use the Nxf1 export pathway (Fig. 1A), selective repression of Nef expression may require a different mechanism than that outlined by Marsh et al. Inhibition of Nef expression was reported to be associated with the accumulation of nef mRNA in cytoplasmic granules that co-stained with markers of stress granules (SGs); these observations led Henao-Mejia et al. to suggest that reduced Nef synthesis was due to sequestration in these bodies. At present, it is unclear whether the granules characterized by Henao-Mejia et al. are similar or distinct from those formed by Sam68ΔC and incompletely spliced HIV-1 RNAs and whether Sam68ΔC inhibition of Nef synthesis is dependent upon their integrity. The two studies suggest that, while the route different RNAs take to repressive sites can differ (the Exportin1 pathway for Rev-dependent RNAs versus the Nxf1 pathway for nef mRNA), a similar mechanism may underlie repression of HIV-1 structural protein and nef mRNAs by Sam68ΔC. However, whether the mechanism is simple sequestration in SGs or something more complex remains to be determined. This is based on the observation of Marsh et al. that RRE-containing RNAs are still repressed upon dispersal of Sam68ΔC granules, although dispersion into functional "nano" granules cannot be dismissed and should be investigated. In addition, ongoing studies (Marsh and Cochrane, unpublished) showing that Sam68ΔC-induced granules contain mRNAs whose expression is not repressed suggest that sequestration to such granules alone is insufficient to explain translational repression. Consequently, additional experiments are needed to assess whether common or distinct mechanisms underlie repression of HIV-1 structural protein and nef mRNAs by Sam68ΔC.

Bottom Line: Two recent publications have explored the mechanisms by which a mutant of the host protein Sam68 blocks HIV-1 structural protein synthesis and expands its activity to encompass Nef.Although the two studies propose different mechanisms for the responses observed, it is possible that a common activity is responsible.Understanding how this Sam68 mutant discriminates among the multiple viral mRNAs promises to reveal unique properties of HIV-1 RNA metabolism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. alan.cochrane@utoronto.ca

ABSTRACT
Two recent publications have explored the mechanisms by which a mutant of the host protein Sam68 blocks HIV-1 structural protein synthesis and expands its activity to encompass Nef. Although the two studies propose different mechanisms for the responses observed, it is possible that a common activity is responsible. Understanding how this Sam68 mutant discriminates among the multiple viral mRNAs promises to reveal unique properties of HIV-1 RNA metabolism.

Show MeSH