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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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FRET measurement of Rev oligomer formation.(A) Expression level and trans-activation capacity of Rev-CFP and Rev-YFP fusion proteins. COS cells were transiently cotransfected with the Rev reporter plasmid pGPV-RRE and the indicated trans-activator constructs. At 24 h post-transfection Rev-dependent expression of HIV-1 structural proteins p55Gag and p24Gag, the respective Rev trans-activators and actin (gel loading control) was detected by Western blot analysis using specific antibodies. (B) Representative FACS plots illustrating the percentage of FRET positive cells. COS cells were transiently transfected with expression vectors for control CFP and RevWT-YFP, or the indicated combinations of Rev donor and acceptor constructs. To provide RRE RNA the plasmid pGPV-RRE was included in each transfection. At 24 h post-transfection cells were harvested and analyzed by flow cytometry for Rev:Rev interaction, represented as FRET positive cells. (C) Mean percentage of FRET-positive cells (adjusted to the background) determined in FRET-FACS experiments.
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pone-0038305-g004: FRET measurement of Rev oligomer formation.(A) Expression level and trans-activation capacity of Rev-CFP and Rev-YFP fusion proteins. COS cells were transiently cotransfected with the Rev reporter plasmid pGPV-RRE and the indicated trans-activator constructs. At 24 h post-transfection Rev-dependent expression of HIV-1 structural proteins p55Gag and p24Gag, the respective Rev trans-activators and actin (gel loading control) was detected by Western blot analysis using specific antibodies. (B) Representative FACS plots illustrating the percentage of FRET positive cells. COS cells were transiently transfected with expression vectors for control CFP and RevWT-YFP, or the indicated combinations of Rev donor and acceptor constructs. To provide RRE RNA the plasmid pGPV-RRE was included in each transfection. At 24 h post-transfection cells were harvested and analyzed by flow cytometry for Rev:Rev interaction, represented as FRET positive cells. (C) Mean percentage of FRET-positive cells (adjusted to the background) determined in FRET-FACS experiments.

Mentions: To directly confirm leucine zipper-dependent interaction of Rev in transfected cells we employed a flow cytometry-based FRET assay system [66]. COS cells were transiently transfected with vectors encoding Rev fusion proteins that were tagged at their carboxy terminus either with a donor or an acceptor fluorophore (CFP or YFP, respectively). The transfection protocol also included the GPV-RRE reporter vector, that contains the HIV-1 gag-pol gene and expresses Gag proteins in a Rev-dependent fashion [67]. At 24 h post-transfection, Western blot analyses revealed the expected Rev phenotypes. Significant amounts of Gag proteins were only detectable in the cultures in which either RevWT or ZipRevSLT40 proteins were expressed, but not in case of RevSLT40 (Figure 4A). FACS-FRET analysis on cell cultures which were transfected in parallel revealed that coexpression of CFP and YFP labelled RevWT resulted in 80.3% of FRET positive cells which fell within the background adjusted gate, indicating pronounced Rev:Rev interaction (Figure 4B and 4C). As expected, the RevSLT40 mutant did not produce a significant FRET signal (2.8%), while expression of ZipRevSLT40 yielded 41.9% of FRET positive cells.


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)

FRET measurement of Rev oligomer formation.(A) Expression level and trans-activation capacity of Rev-CFP and Rev-YFP fusion proteins. COS cells were transiently cotransfected with the Rev reporter plasmid pGPV-RRE and the indicated trans-activator constructs. At 24 h post-transfection Rev-dependent expression of HIV-1 structural proteins p55Gag and p24Gag, the respective Rev trans-activators and actin (gel loading control) was detected by Western blot analysis using specific antibodies. (B) Representative FACS plots illustrating the percentage of FRET positive cells. COS cells were transiently transfected with expression vectors for control CFP and RevWT-YFP, or the indicated combinations of Rev donor and acceptor constructs. To provide RRE RNA the plasmid pGPV-RRE was included in each transfection. At 24 h post-transfection cells were harvested and analyzed by flow cytometry for Rev:Rev interaction, represented as FRET positive cells. (C) Mean percentage of FRET-positive cells (adjusted to the background) determined in FRET-FACS experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366918&req=5

pone-0038305-g004: FRET measurement of Rev oligomer formation.(A) Expression level and trans-activation capacity of Rev-CFP and Rev-YFP fusion proteins. COS cells were transiently cotransfected with the Rev reporter plasmid pGPV-RRE and the indicated trans-activator constructs. At 24 h post-transfection Rev-dependent expression of HIV-1 structural proteins p55Gag and p24Gag, the respective Rev trans-activators and actin (gel loading control) was detected by Western blot analysis using specific antibodies. (B) Representative FACS plots illustrating the percentage of FRET positive cells. COS cells were transiently transfected with expression vectors for control CFP and RevWT-YFP, or the indicated combinations of Rev donor and acceptor constructs. To provide RRE RNA the plasmid pGPV-RRE was included in each transfection. At 24 h post-transfection cells were harvested and analyzed by flow cytometry for Rev:Rev interaction, represented as FRET positive cells. (C) Mean percentage of FRET-positive cells (adjusted to the background) determined in FRET-FACS experiments.
Mentions: To directly confirm leucine zipper-dependent interaction of Rev in transfected cells we employed a flow cytometry-based FRET assay system [66]. COS cells were transiently transfected with vectors encoding Rev fusion proteins that were tagged at their carboxy terminus either with a donor or an acceptor fluorophore (CFP or YFP, respectively). The transfection protocol also included the GPV-RRE reporter vector, that contains the HIV-1 gag-pol gene and expresses Gag proteins in a Rev-dependent fashion [67]. At 24 h post-transfection, Western blot analyses revealed the expected Rev phenotypes. Significant amounts of Gag proteins were only detectable in the cultures in which either RevWT or ZipRevSLT40 proteins were expressed, but not in case of RevSLT40 (Figure 4A). FACS-FRET analysis on cell cultures which were transfected in parallel revealed that coexpression of CFP and YFP labelled RevWT resulted in 80.3% of FRET positive cells which fell within the background adjusted gate, indicating pronounced Rev:Rev interaction (Figure 4B and 4C). As expected, the RevSLT40 mutant did not produce a significant FRET signal (2.8%), while expression of ZipRevSLT40 yielded 41.9% of FRET positive cells.

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