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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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Analysis of Rev-mediated nuclear RNA export.(A) Schematic representation of the nuclease S1 protection assay. The Rev reporter, the end-labelled input probe and the protected sequences, recognizing unspliced and spliced RNA are depicted. (B) Nucleocytoplasmic distribution of Rev-regulated unspliced and spliced RNA determined by nuclease S1 assay. COS cells were transiently cotransfected with Rev reporter DNA pDM128/CMV and the indicated Rev expression vectors. Nuclear and cytoplasmic RNA was isolated at 48 h post-transfection. Unspliced and spliced reporter-derived RNA was detected by autoradiography using the 32P-labeled input probe. (N, nuclear RNA; C, cytoplasmic RNA). (C) Quantification of Rev-regulated cytoplasmic and nuclear viral RNA. HeLa cells were transiently cotransfected with pHXB2Δrev and the indicated Rev constructs. At 48 h post-transfection nuclear and cytoplasmic RNAs were isolated and the accumulation of Rev-regulated unspliced gag RNA was quantified by real-time PCR. Total viral copy numbers were adjusted to the endogenous gapdh mRNA level in each sample.
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pone-0038305-g008: Analysis of Rev-mediated nuclear RNA export.(A) Schematic representation of the nuclease S1 protection assay. The Rev reporter, the end-labelled input probe and the protected sequences, recognizing unspliced and spliced RNA are depicted. (B) Nucleocytoplasmic distribution of Rev-regulated unspliced and spliced RNA determined by nuclease S1 assay. COS cells were transiently cotransfected with Rev reporter DNA pDM128/CMV and the indicated Rev expression vectors. Nuclear and cytoplasmic RNA was isolated at 48 h post-transfection. Unspliced and spliced reporter-derived RNA was detected by autoradiography using the 32P-labeled input probe. (N, nuclear RNA; C, cytoplasmic RNA). (C) Quantification of Rev-regulated cytoplasmic and nuclear viral RNA. HeLa cells were transiently cotransfected with pHXB2Δrev and the indicated Rev constructs. At 48 h post-transfection nuclear and cytoplasmic RNAs were isolated and the accumulation of Rev-regulated unspliced gag RNA was quantified by real-time PCR. Total viral copy numbers were adjusted to the endogenous gapdh mRNA level in each sample.

Mentions: Rev-dependent nucleocytoplasmic distribution of RRE-containing mRNA was next monitored by using two different experimental designs. First, COS cells were transiently cotransfected with the Rev-responsive reporter construct pDM128/CMV and various Rev expression plasmids. At 48 h post-transfection nuclear and cytoplasmic RNA was collected from the transfected cell cultures and analyzed by quantitative S1 nuclease protection analyses. The endlabelled input probe allowed its discrimination from unspliced and spliced reporter mRNA (depicted in Figure 8A). Inspection of the obtained data revealed that the oligomerization-defective mutant RevSLT40 failed to mediate efficient cytoplasmic accumulation of unspliced mRNA (Figure 8B). Obviously, nuclear export of these RRE-containing transcripts was restored when the ZipRevSLT40 fusion protein was coexpressed, indicating that oligomer-formation of Rev is required for the translocation of Rev-regulated transcripts across the nuclear envelope.


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)

Analysis of Rev-mediated nuclear RNA export.(A) Schematic representation of the nuclease S1 protection assay. The Rev reporter, the end-labelled input probe and the protected sequences, recognizing unspliced and spliced RNA are depicted. (B) Nucleocytoplasmic distribution of Rev-regulated unspliced and spliced RNA determined by nuclease S1 assay. COS cells were transiently cotransfected with Rev reporter DNA pDM128/CMV and the indicated Rev expression vectors. Nuclear and cytoplasmic RNA was isolated at 48 h post-transfection. Unspliced and spliced reporter-derived RNA was detected by autoradiography using the 32P-labeled input probe. (N, nuclear RNA; C, cytoplasmic RNA). (C) Quantification of Rev-regulated cytoplasmic and nuclear viral RNA. HeLa cells were transiently cotransfected with pHXB2Δrev and the indicated Rev constructs. At 48 h post-transfection nuclear and cytoplasmic RNAs were isolated and the accumulation of Rev-regulated unspliced gag RNA was quantified by real-time PCR. Total viral copy numbers were adjusted to the endogenous gapdh mRNA level in each sample.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366918&req=5

pone-0038305-g008: Analysis of Rev-mediated nuclear RNA export.(A) Schematic representation of the nuclease S1 protection assay. The Rev reporter, the end-labelled input probe and the protected sequences, recognizing unspliced and spliced RNA are depicted. (B) Nucleocytoplasmic distribution of Rev-regulated unspliced and spliced RNA determined by nuclease S1 assay. COS cells were transiently cotransfected with Rev reporter DNA pDM128/CMV and the indicated Rev expression vectors. Nuclear and cytoplasmic RNA was isolated at 48 h post-transfection. Unspliced and spliced reporter-derived RNA was detected by autoradiography using the 32P-labeled input probe. (N, nuclear RNA; C, cytoplasmic RNA). (C) Quantification of Rev-regulated cytoplasmic and nuclear viral RNA. HeLa cells were transiently cotransfected with pHXB2Δrev and the indicated Rev constructs. At 48 h post-transfection nuclear and cytoplasmic RNAs were isolated and the accumulation of Rev-regulated unspliced gag RNA was quantified by real-time PCR. Total viral copy numbers were adjusted to the endogenous gapdh mRNA level in each sample.
Mentions: Rev-dependent nucleocytoplasmic distribution of RRE-containing mRNA was next monitored by using two different experimental designs. First, COS cells were transiently cotransfected with the Rev-responsive reporter construct pDM128/CMV and various Rev expression plasmids. At 48 h post-transfection nuclear and cytoplasmic RNA was collected from the transfected cell cultures and analyzed by quantitative S1 nuclease protection analyses. The endlabelled input probe allowed its discrimination from unspliced and spliced reporter mRNA (depicted in Figure 8A). Inspection of the obtained data revealed that the oligomerization-defective mutant RevSLT40 failed to mediate efficient cytoplasmic accumulation of unspliced mRNA (Figure 8B). Obviously, nuclear export of these RRE-containing transcripts was restored when the ZipRevSLT40 fusion protein was coexpressed, indicating that oligomer-formation of Rev is required for the translocation of Rev-regulated transcripts across the nuclear envelope.

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