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Formation of trans-activation competent HIV-1 Rev:RRE complexes requires the recruitment of multiple protein activation domains.

Hoffmann D, Schwarck D, Banning C, Brenner M, Mariyanna L, Krepstakies M, Schindler M, Millar DP, Hauber J - PLoS ONE (2012)

Bottom Line: In contrast, however, the formation of Rev oligomers on the RRE is a precondition to trans-activation by directly affecting the nuclear export of Rev-regulated mRNA.Moreover, experimental evidence is provided showing that at least two protein activation domains are required for the formation of trans-activation competent Rev:RRE complexes.The presented data further refine the model of Rev trans-activation by directly demonstrating that Rev oligomerization on the RRE, thereby recruiting at least two protein activation domains, is required for nuclear export of unspliced and incompletely spliced viral RNA.

View Article: PubMed Central - PubMed

Affiliation: Heinrich Pette Institute - Leibniz Institute for Experimental Virology, Hamburg, Germany.

ABSTRACT
The HIV-1 Rev trans-activator is a nucleocytoplasmic shuttle protein that is essential for virus replication. Rev directly binds to unspliced and incompletely spliced viral RNA via the cis-acting Rev Response Element (RRE) sequence. Subsequently, Rev oligomerizes cooperatively and interacts with the cellular nuclear export receptor CRM1. In addition to mediating nuclear RNA export, Rev also affects the stability, translation and packaging of Rev-bound viral transcripts. Although it is established that Rev function requires the multimeric assembly of Rev molecules on the RRE, relatively little is known about how many Rev monomers are sufficient to form a trans-activation competent Rev:RRE complex, or which specific activity of Rev is affected by its oligomerization. We here analyzed by functional studies how homooligomer formation of Rev affects the trans-activation capacity of this essential HIV-1 regulatory protein. In a gain-of-function approach, we fused various heterologous dimerization domains to an otherwise oligomerization-defective Rev mutant and were able to demonstrate that oligomerization of Rev is not required per se for the nuclear export of this viral trans-activator. In contrast, however, the formation of Rev oligomers on the RRE is a precondition to trans-activation by directly affecting the nuclear export of Rev-regulated mRNA. Moreover, experimental evidence is provided showing that at least two protein activation domains are required for the formation of trans-activation competent Rev:RRE complexes. The presented data further refine the model of Rev trans-activation by directly demonstrating that Rev oligomerization on the RRE, thereby recruiting at least two protein activation domains, is required for nuclear export of unspliced and incompletely spliced viral RNA.

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In vivo RNA binding by Rev mutants.(A) Schematic representation of the pSLIIB/CAT reporter used to analyze Rev:RRE RNA binding in vivo. (B) In vivo RRE RNA-binding phenotypes of various trans-activators are shown. HeLa cells were transiently cotransfected with the Rev reporter construct pSLIIB/CAT, pBC12/CMV/β-Gal (internal control) and the indicated Tat-Rev fusion constructs. At 48 h post-transfection, CAT and β-Gal expression was analysed by ELISA. CAT values were adjusted for transfection efficiency to the β-Gal level in each culture. Data are expressed as a percentage of wildtype Rev activity (set to 100%).
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pone-0038305-g002: In vivo RNA binding by Rev mutants.(A) Schematic representation of the pSLIIB/CAT reporter used to analyze Rev:RRE RNA binding in vivo. (B) In vivo RRE RNA-binding phenotypes of various trans-activators are shown. HeLa cells were transiently cotransfected with the Rev reporter construct pSLIIB/CAT, pBC12/CMV/β-Gal (internal control) and the indicated Tat-Rev fusion constructs. At 48 h post-transfection, CAT and β-Gal expression was analysed by ELISA. CAT values were adjusted for transfection efficiency to the β-Gal level in each culture. Data are expressed as a percentage of wildtype Rev activity (set to 100%).

Mentions: We first employed an established reporter gene-based in vivo RNA binding assay [55], [64] to confirm that the respective ZipRevSLT40 protein is able to recognize RRE RNA. The pSLIIB/CAT reporter construct contains the CAT gene under the transcriptional control of the HIV-1 LTR promoter (depicted in Figure 2A). The wildtype TAR element, which is the promoter-proximal RNA target sequence of the HIV-1 Tat transcriptional trans-activator, is replaced by a sequence encoding the RRE-derived SLIIB high-affinity Rev binding site. This promoter is only activated by Tat-Rev fusion proteins and is not responsive to Tat or Rev alone [64]. Thus, the quantity of CAT produced gives an indication of the RNA binding ability of a given Rev mutant.


Formation of trans-activation competent HIV-1 Rev:RRE complexes requires the recruitment of multiple protein activation domains.

Hoffmann D, Schwarck D, Banning C, Brenner M, Mariyanna L, Krepstakies M, Schindler M, Millar DP, Hauber J - PLoS ONE (2012)

In vivo RNA binding by Rev mutants.(A) Schematic representation of the pSLIIB/CAT reporter used to analyze Rev:RRE RNA binding in vivo. (B) In vivo RRE RNA-binding phenotypes of various trans-activators are shown. HeLa cells were transiently cotransfected with the Rev reporter construct pSLIIB/CAT, pBC12/CMV/β-Gal (internal control) and the indicated Tat-Rev fusion constructs. At 48 h post-transfection, CAT and β-Gal expression was analysed by ELISA. CAT values were adjusted for transfection efficiency to the β-Gal level in each culture. Data are expressed as a percentage of wildtype Rev activity (set to 100%).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366918&req=5

pone-0038305-g002: In vivo RNA binding by Rev mutants.(A) Schematic representation of the pSLIIB/CAT reporter used to analyze Rev:RRE RNA binding in vivo. (B) In vivo RRE RNA-binding phenotypes of various trans-activators are shown. HeLa cells were transiently cotransfected with the Rev reporter construct pSLIIB/CAT, pBC12/CMV/β-Gal (internal control) and the indicated Tat-Rev fusion constructs. At 48 h post-transfection, CAT and β-Gal expression was analysed by ELISA. CAT values were adjusted for transfection efficiency to the β-Gal level in each culture. Data are expressed as a percentage of wildtype Rev activity (set to 100%).
Mentions: We first employed an established reporter gene-based in vivo RNA binding assay [55], [64] to confirm that the respective ZipRevSLT40 protein is able to recognize RRE RNA. The pSLIIB/CAT reporter construct contains the CAT gene under the transcriptional control of the HIV-1 LTR promoter (depicted in Figure 2A). The wildtype TAR element, which is the promoter-proximal RNA target sequence of the HIV-1 Tat transcriptional trans-activator, is replaced by a sequence encoding the RRE-derived SLIIB high-affinity Rev binding site. This promoter is only activated by Tat-Rev fusion proteins and is not responsive to Tat or Rev alone [64]. Thus, the quantity of CAT produced gives an indication of the RNA binding ability of a given Rev mutant.

Bottom Line: In contrast, however, the formation of Rev oligomers on the RRE is a precondition to trans-activation by directly affecting the nuclear export of Rev-regulated mRNA.Moreover, experimental evidence is provided showing that at least two protein activation domains are required for the formation of trans-activation competent Rev:RRE complexes.The presented data further refine the model of Rev trans-activation by directly demonstrating that Rev oligomerization on the RRE, thereby recruiting at least two protein activation domains, is required for nuclear export of unspliced and incompletely spliced viral RNA.

View Article: PubMed Central - PubMed

Affiliation: Heinrich Pette Institute - Leibniz Institute for Experimental Virology, Hamburg, Germany.

ABSTRACT
The HIV-1 Rev trans-activator is a nucleocytoplasmic shuttle protein that is essential for virus replication. Rev directly binds to unspliced and incompletely spliced viral RNA via the cis-acting Rev Response Element (RRE) sequence. Subsequently, Rev oligomerizes cooperatively and interacts with the cellular nuclear export receptor CRM1. In addition to mediating nuclear RNA export, Rev also affects the stability, translation and packaging of Rev-bound viral transcripts. Although it is established that Rev function requires the multimeric assembly of Rev molecules on the RRE, relatively little is known about how many Rev monomers are sufficient to form a trans-activation competent Rev:RRE complex, or which specific activity of Rev is affected by its oligomerization. We here analyzed by functional studies how homooligomer formation of Rev affects the trans-activation capacity of this essential HIV-1 regulatory protein. In a gain-of-function approach, we fused various heterologous dimerization domains to an otherwise oligomerization-defective Rev mutant and were able to demonstrate that oligomerization of Rev is not required per se for the nuclear export of this viral trans-activator. In contrast, however, the formation of Rev oligomers on the RRE is a precondition to trans-activation by directly affecting the nuclear export of Rev-regulated mRNA. Moreover, experimental evidence is provided showing that at least two protein activation domains are required for the formation of trans-activation competent Rev:RRE complexes. The presented data further refine the model of Rev trans-activation by directly demonstrating that Rev oligomerization on the RRE, thereby recruiting at least two protein activation domains, is required for nuclear export of unspliced and incompletely spliced viral RNA.

Show MeSH
Related in: MedlinePlus