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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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Nucleocytoplasmic shuttling of Rev.Standard interspecies heterokaryon fusion assay for detection of protein nuclear export. HeLa cells were transiently transfected with the indicated Rev expression plasmids and fused at 24 h post-transfection with untransfected NIH3T3 cells (indicated by an asterisk). CRM1-mediated protein shuttling was blocked by treatment of the cultures with leptomycin B (LMB). Localization and nuclear export of Rev proteins was visualized by indirect immunofluorescence microscopy using specific anti-Rev antibody (red label; panel b, e, h, k, n, q, t). Nuclei were stained with Hoechst 33258 (blue label; panel a, d, g, j, m, p, s) and merged with the fluorescence and bright light pictures (panel c, f, i, l, o, r, u).
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pone-0038305-g007: Nucleocytoplasmic shuttling of Rev.Standard interspecies heterokaryon fusion assay for detection of protein nuclear export. HeLa cells were transiently transfected with the indicated Rev expression plasmids and fused at 24 h post-transfection with untransfected NIH3T3 cells (indicated by an asterisk). CRM1-mediated protein shuttling was blocked by treatment of the cultures with leptomycin B (LMB). Localization and nuclear export of Rev proteins was visualized by indirect immunofluorescence microscopy using specific anti-Rev antibody (red label; panel b, e, h, k, n, q, t). Nuclei were stained with Hoechst 33258 (blue label; panel a, d, g, j, m, p, s) and merged with the fluorescence and bright light pictures (panel c, f, i, l, o, r, u).

Mentions: To directly analyze the nuclear export ability of RevSLT40 and ZipRevSLT40, standard heterokaryon assays were performed, in which transiently transfected human HeLa cells (expressing the various Rev proteins) were fused with untransfected mouse NIH 3T3 cells to form heterokaryons. Fixed cells were analyzed by indirect immunofluorescence microscopy using Rev-specific antibodies. The respective cultures were also treated with Hoechst 33258 dye, which allows discrimination of human and mouse nuclei (the latter are characterized by a typical speckle pattern). Clearly, RevWT protein was able to migrate from the human to the mouse nucleus in these experiments (Figure 7, panel a–c). As expected, exposure of the cell culture to leptomycin B (LMB), a highly specific small-molecular weight inhibitor of the CRM1 nuclear export receptor [70], [71], abolished Rev’s ability to exit the human nucleus (Figure 7, panel d–f). A comparable result was also observed in a control experiment when the export-deficient mutant RevM10 was analyzed (Figure 7, panel s–u). Interestingly, both the oligomerization- and trans-activation-deficient mutant RevSLT40, as well as the ZipRevSLT40 protein, displayed comparable CRM1-dependent nuclear export activities in this type of assay (Figure 7, panel g–l and panel m–r, respectively). These data suggested that, in absence of RRE RNA, oligomerization of Rev is not required for nuclear export of Rev proteins via the CRM1-pathway.


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)

Nucleocytoplasmic shuttling of Rev.Standard interspecies heterokaryon fusion assay for detection of protein nuclear export. HeLa cells were transiently transfected with the indicated Rev expression plasmids and fused at 24 h post-transfection with untransfected NIH3T3 cells (indicated by an asterisk). CRM1-mediated protein shuttling was blocked by treatment of the cultures with leptomycin B (LMB). Localization and nuclear export of Rev proteins was visualized by indirect immunofluorescence microscopy using specific anti-Rev antibody (red label; panel b, e, h, k, n, q, t). Nuclei were stained with Hoechst 33258 (blue label; panel a, d, g, j, m, p, s) and merged with the fluorescence and bright light pictures (panel c, f, i, l, o, r, u).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366918&req=5

pone-0038305-g007: Nucleocytoplasmic shuttling of Rev.Standard interspecies heterokaryon fusion assay for detection of protein nuclear export. HeLa cells were transiently transfected with the indicated Rev expression plasmids and fused at 24 h post-transfection with untransfected NIH3T3 cells (indicated by an asterisk). CRM1-mediated protein shuttling was blocked by treatment of the cultures with leptomycin B (LMB). Localization and nuclear export of Rev proteins was visualized by indirect immunofluorescence microscopy using specific anti-Rev antibody (red label; panel b, e, h, k, n, q, t). Nuclei were stained with Hoechst 33258 (blue label; panel a, d, g, j, m, p, s) and merged with the fluorescence and bright light pictures (panel c, f, i, l, o, r, u).
Mentions: To directly analyze the nuclear export ability of RevSLT40 and ZipRevSLT40, standard heterokaryon assays were performed, in which transiently transfected human HeLa cells (expressing the various Rev proteins) were fused with untransfected mouse NIH 3T3 cells to form heterokaryons. Fixed cells were analyzed by indirect immunofluorescence microscopy using Rev-specific antibodies. The respective cultures were also treated with Hoechst 33258 dye, which allows discrimination of human and mouse nuclei (the latter are characterized by a typical speckle pattern). Clearly, RevWT protein was able to migrate from the human to the mouse nucleus in these experiments (Figure 7, panel a–c). As expected, exposure of the cell culture to leptomycin B (LMB), a highly specific small-molecular weight inhibitor of the CRM1 nuclear export receptor [70], [71], abolished Rev’s ability to exit the human nucleus (Figure 7, panel d–f). A comparable result was also observed in a control experiment when the export-deficient mutant RevM10 was analyzed (Figure 7, panel s–u). Interestingly, both the oligomerization- and trans-activation-deficient mutant RevSLT40, as well as the ZipRevSLT40 protein, displayed comparable CRM1-dependent nuclear export activities in this type of assay (Figure 7, panel g–l and panel m–r, respectively). These data suggested that, in absence of RRE RNA, oligomerization of Rev is not required for nuclear export of Rev proteins via the CRM1-pathway.

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