Differential effects of Tra2β isoforms on HIV-1 RNA processing and expression
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POSTER PRESENTATION Open Access Differential effects of Tra2b isoforms on HIV-1 RNA processing and expression Craig Platt, Maria Calimano, Josip Nemet, Jodi Bubenik, Alan Cochrane * From Frontiers of Retrovirology: Complex retroviruses, retroelements and their hosts Cambridge, UK. 16-18 September 2013 Background Balanced processing of HIV-1 RNA is critical to virus replication and is regulated by host factors such as SR proteins. To examine the role of the SR-related proteins Tra2a and Tra2b in regulating viral gene expression, we used both overexpression and depletion analysis to examine how these factors impacted HIV-1 RNA spli- cing, transport and expression and used mutation to identify the protein domains involved. Materials and methods HEK 293/293T cells were transfected with plasmids expressing HIV-1 provirus and with vectors expressing Tra2a, Tra2b, or mutants thereof. Effects on viral gene expression and RNA processing were monitored by wes- tern/northern blot, in situ hybridization, and RT-PCR. To examine effects of Tra2b depletion, cells were trans- duced with lentivirus expressing control or anti-Tra2b shRNAs and similar analyses used to monitor changes in HIV-1 RNA processing and expression. Results Overexpression of either Tra2a or Tra2b results in a marked reduction in HIV-1 Gag/Env expression, an effect associated with changes in HIV-1 RNA accumula- tion and a block to export of HIV-1 genomic RNA. Mutagenesis to define the domains critical for the inhi- bitory activity revealed that a natural isoform of Tra2b (Tra2b3), lacking the N-terminal RS domain, also sup- pressed HIV-1 expression but had very different effects on viral RNA processing at both the level of accumula- tion of the various viral RNAs (unspliced, singly spliced and multiply spliced) as well as splice site usage. In con- trast, variants lacking the C-terminal RS domain or point mutants that disrupt RNA binding had no effect. Tests to define elements that mediate the response determined that the ESE3 and ESS$ within the terminal exon of HIV-1 are not required for the effects observed. The functional differences between the Tra2b isoforms were also observed in the context of another RNA sub- strate indicating that these factors have distinct func- tions within the cell. Finally, we demonstrate that Tra2b depletion results in a selective reduction in HIV-1 Env expression that is correlated with decreased accumula- tion of the corresponding viral RNA. Conclusions Together, these findings indicate that Tra2a/b can play important roles in regulating HIV-1 RNA metabolism and expression, suggesting that modulation of its activity could be used to suppress virus replication. Further- more, we demonstrate that the different isoforms of Tra2b (b1 and b3), while inducing similar reductions in HIV-1 Gag and Env expression appear to achieve this end by modulating viral RNA processing in different ways. Published: 19 September 2013 doi:10.1186/1742-4690-10-S1-P17 Cite this article as: Platt et al.: Differential effects of Tra2b isoforms on HIV-1 RNA processing and expression. Retrovirology 2013 10(Suppl 1):P17. Molecular Genetics, University of Toronto, Toronto, Ontario, Canada Platt et al. Retrovirology 2013, 10(Suppl 1):P17 http://www.retrovirology.com/content/10/S1/P17 © 2013 Platt et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Transcript of Differential effects of Tra2β isoforms on HIV-1 RNA processing and expression
POSTER PRESENTATION Open Access
Differential effects of Tra2b isoforms on HIV-1RNA processing and expressionCraig Platt, Maria Calimano, Josip Nemet, Jodi Bubenik, Alan Cochrane*
From Frontiers of Retrovirology: Complex retroviruses, retroelements and their hostsCambridge, UK. 16-18 September 2013
BackgroundBalanced processing of HIV-1 RNA is critical to virusreplication and is regulated by host factors such as SRproteins. To examine the role of the SR-related proteinsTra2a and Tra2b in regulating viral gene expression, weused both overexpression and depletion analysis toexamine how these factors impacted HIV-1 RNA spli-cing, transport and expression and used mutation toidentify the protein domains involved.
Materials and methodsHEK 293/293T cells were transfected with plasmidsexpressing HIV-1 provirus and with vectors expressingTra2a, Tra2b, or mutants thereof. Effects on viral geneexpression and RNA processing were monitored by wes-tern/northern blot, in situ hybridization, and RT-PCR.To examine effects of Tra2b depletion, cells were trans-duced with lentivirus expressing control or anti-Tra2bshRNAs and similar analyses used to monitor changesin HIV-1 RNA processing and expression.
ResultsOverexpression of either Tra2a or Tra2b results in amarked reduction in HIV-1 Gag/Env expression, aneffect associated with changes in HIV-1 RNA accumula-tion and a block to export of HIV-1 genomic RNA.Mutagenesis to define the domains critical for the inhi-bitory activity revealed that a natural isoform of Tra2b(Tra2b3), lacking the N-terminal RS domain, also sup-pressed HIV-1 expression but had very different effectson viral RNA processing at both the level of accumula-tion of the various viral RNAs (unspliced, singly splicedand multiply spliced) as well as splice site usage. In con-trast, variants lacking the C-terminal RS domain orpoint mutants that disrupt RNA binding had no effect.
Tests to define elements that mediate the responsedetermined that the ESE3 and ESS$ within the terminalexon of HIV-1 are not required for the effects observed.The functional differences between the Tra2b isoformswere also observed in the context of another RNA sub-strate indicating that these factors have distinct func-tions within the cell. Finally, we demonstrate that Tra2bdepletion results in a selective reduction in HIV-1 Envexpression that is correlated with decreased accumula-tion of the corresponding viral RNA.
ConclusionsTogether, these findings indicate that Tra2a/b can playimportant roles in regulating HIV-1 RNA metabolismand expression, suggesting that modulation of its activitycould be used to suppress virus replication. Further-more, we demonstrate that the different isoforms ofTra2b (b1 and b3), while inducing similar reductions inHIV-1 Gag and Env expression appear to achieve thisend by modulating viral RNA processing in differentways.
Published: 19 September 2013
doi:10.1186/1742-4690-10-S1-P17Cite this article as: Platt et al.: Differential effects of Tra2b isoforms onHIV-1 RNA processing and expression. Retrovirology 2013 10(Suppl 1):P17.
Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
Platt et al. Retrovirology 2013, 10(Suppl 1):P17http://www.retrovirology.com/content/10/S1/P17
© 2013 Platt et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.
