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Differential Effects of Tra2ß Isoforms on HIV-1 RNA Processing and Expression.

Platt C, Calimano M, Nemet J, Bubenik J, Cochrane A - PLoS ONE (2015)

Bottom Line: In this report, we demonstrate that overexpression of either Tra2α or Tra2β results in a marked reduction in HIV-1 Gag/Env expression, an effect associated with changes in HIV-1 RNA accumulation, altered viral splice site usage, and a block to export of HIV-1 genomic RNA.A natural isoform of Tra2β (Tra2ß3), lacking the N-terminal RS domain, also suppressed HIV-1 expression but had different effects on viral RNA processing.Finally, we demonstrate that Tra2ß depletion results in a selective reduction in HIV-1 Env expression as well as an increase in multiply spliced viral RNA.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Balanced processing of HIV-1 RNA is critical to virus replication and is regulated by host factors. In this report, we demonstrate that overexpression of either Tra2α or Tra2β results in a marked reduction in HIV-1 Gag/Env expression, an effect associated with changes in HIV-1 RNA accumulation, altered viral splice site usage, and a block to export of HIV-1 genomic RNA. A natural isoform of Tra2β (Tra2ß3), lacking the N-terminal RS domain, also suppressed HIV-1 expression but had different effects on viral RNA processing. The functional differences between the Tra2β isoforms were also observed in the context of another RNA substrate indicating that these factors have distinct functions within the cell. Finally, we demonstrate that Tra2ß depletion results in a selective reduction in HIV-1 Env expression as well as an increase in multiply spliced viral RNA. Together, the findings indicate that Tra2α/β can play important roles in regulating HIV-1 RNA metabolism and expression.

No MeSH data available.


Related in: MedlinePlus

Modulation of HIV-1 US RNA Transport by Tra2β/βΔN Overexpression.293 cells were transfected with pHxb2 R-/RI- and plasmids expressing GFP (GFP) or GFP—tagged Tra2β (GFP Tra2β), Tra2βΔN (GFP Tra2βΔN), Tra2βΔC (GFP Tra2βΔC), or Tra2βffdd (GFP Tra2βffdd). Cells were fixed 48 h post-transfection and processed for localization of HIV-1 US RNA and GFP-tagged protein (GFP) as detailed in “Materials and Methods”. Nuclei were detected by staining with DAPI.
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pone.0125315.g005: Modulation of HIV-1 US RNA Transport by Tra2β/βΔN Overexpression.293 cells were transfected with pHxb2 R-/RI- and plasmids expressing GFP (GFP) or GFP—tagged Tra2β (GFP Tra2β), Tra2βΔN (GFP Tra2βΔN), Tra2βΔC (GFP Tra2βΔC), or Tra2βffdd (GFP Tra2βffdd). Cells were fixed 48 h post-transfection and processed for localization of HIV-1 US RNA and GFP-tagged protein (GFP) as detailed in “Materials and Methods”. Nuclei were detected by staining with DAPI.

Mentions: The absence of HIV-1 Gag and Env expression in the presence of either Tra2β or Tra2βΔN, despite the presence of significant levels of the corresponding viral (US and SS) RNAs (Fig 2B), suggested the possibility that these factors might act to alter viral RNA transport. To address this hypothesis, in situ hybridization was used to examine viral US RNA subcellular localization in response to overexpression of Tra2β or its variants. As shown in Fig 5, co-transfection of HIV-1 proviral DNA with control vector (peGFP) resulted in the majority of the US viral RNA being localized to the cytoplasm. A similar pattern of HIV-1 US RNA distribution was also observed upon co-transfection with Tra2βΔC or Tra2βffdd, neither of which altered HIV-1 RNA processing (Fig 3 and Fig C in S1 File). In contrast, co-transfection with either Tra2β or Tra2βΔN resulted in the viral US RNA being present predominately in the nucleus, indicative of a block to nuclear export. Similar overexpression studies using GFP fused to SC35, SRp20 or 9G8 did not elicit a similar nuclear sequestration of HIV-1 US RNA (Fig 6), indicating that the response to Tra2β/Tra2βΔN overexpression is not a general response to SR overexpression but is limited to a least a subset of proteins within the SR family. Additional tests (Fig D in S1 File) confirmed expression and the effect of the GFP fusion proteins on HIV-1 gene expression.


Differential Effects of Tra2ß Isoforms on HIV-1 RNA Processing and Expression.

Platt C, Calimano M, Nemet J, Bubenik J, Cochrane A - PLoS ONE (2015)

Modulation of HIV-1 US RNA Transport by Tra2β/βΔN Overexpression.293 cells were transfected with pHxb2 R-/RI- and plasmids expressing GFP (GFP) or GFP—tagged Tra2β (GFP Tra2β), Tra2βΔN (GFP Tra2βΔN), Tra2βΔC (GFP Tra2βΔC), or Tra2βffdd (GFP Tra2βffdd). Cells were fixed 48 h post-transfection and processed for localization of HIV-1 US RNA and GFP-tagged protein (GFP) as detailed in “Materials and Methods”. Nuclei were detected by staining with DAPI.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430212&req=5

pone.0125315.g005: Modulation of HIV-1 US RNA Transport by Tra2β/βΔN Overexpression.293 cells were transfected with pHxb2 R-/RI- and plasmids expressing GFP (GFP) or GFP—tagged Tra2β (GFP Tra2β), Tra2βΔN (GFP Tra2βΔN), Tra2βΔC (GFP Tra2βΔC), or Tra2βffdd (GFP Tra2βffdd). Cells were fixed 48 h post-transfection and processed for localization of HIV-1 US RNA and GFP-tagged protein (GFP) as detailed in “Materials and Methods”. Nuclei were detected by staining with DAPI.
Mentions: The absence of HIV-1 Gag and Env expression in the presence of either Tra2β or Tra2βΔN, despite the presence of significant levels of the corresponding viral (US and SS) RNAs (Fig 2B), suggested the possibility that these factors might act to alter viral RNA transport. To address this hypothesis, in situ hybridization was used to examine viral US RNA subcellular localization in response to overexpression of Tra2β or its variants. As shown in Fig 5, co-transfection of HIV-1 proviral DNA with control vector (peGFP) resulted in the majority of the US viral RNA being localized to the cytoplasm. A similar pattern of HIV-1 US RNA distribution was also observed upon co-transfection with Tra2βΔC or Tra2βffdd, neither of which altered HIV-1 RNA processing (Fig 3 and Fig C in S1 File). In contrast, co-transfection with either Tra2β or Tra2βΔN resulted in the viral US RNA being present predominately in the nucleus, indicative of a block to nuclear export. Similar overexpression studies using GFP fused to SC35, SRp20 or 9G8 did not elicit a similar nuclear sequestration of HIV-1 US RNA (Fig 6), indicating that the response to Tra2β/Tra2βΔN overexpression is not a general response to SR overexpression but is limited to a least a subset of proteins within the SR family. Additional tests (Fig D in S1 File) confirmed expression and the effect of the GFP fusion proteins on HIV-1 gene expression.

Bottom Line: In this report, we demonstrate that overexpression of either Tra2α or Tra2β results in a marked reduction in HIV-1 Gag/Env expression, an effect associated with changes in HIV-1 RNA accumulation, altered viral splice site usage, and a block to export of HIV-1 genomic RNA.A natural isoform of Tra2β (Tra2ß3), lacking the N-terminal RS domain, also suppressed HIV-1 expression but had different effects on viral RNA processing.Finally, we demonstrate that Tra2ß depletion results in a selective reduction in HIV-1 Env expression as well as an increase in multiply spliced viral RNA.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Balanced processing of HIV-1 RNA is critical to virus replication and is regulated by host factors. In this report, we demonstrate that overexpression of either Tra2α or Tra2β results in a marked reduction in HIV-1 Gag/Env expression, an effect associated with changes in HIV-1 RNA accumulation, altered viral splice site usage, and a block to export of HIV-1 genomic RNA. A natural isoform of Tra2β (Tra2ß3), lacking the N-terminal RS domain, also suppressed HIV-1 expression but had different effects on viral RNA processing. The functional differences between the Tra2β isoforms were also observed in the context of another RNA substrate indicating that these factors have distinct functions within the cell. Finally, we demonstrate that Tra2ß depletion results in a selective reduction in HIV-1 Env expression as well as an increase in multiply spliced viral RNA. Together, the findings indicate that Tra2α/β can play important roles in regulating HIV-1 RNA metabolism and expression.

No MeSH data available.


Related in: MedlinePlus