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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

Tra2β Depletion Results in Selective Alterations in HIV-1 RNA Accumulation.HeLa rtTA HIVΔmls cells were transduced with lentiviruses expressing either control (shNT) or shRNA targeting Tra2β (shTra2β). After infection, transduced cells were selected using puromycin for 48–72 h, then HIV-1 provirus expression induced by addition of doxycycline. RNA was subsequently extracted and analyzed (A) by qRT-PCR to examine for changes in HIV-1 US, SS and MS RNA levels or (B) RT-PCR of MS RNAs to examine for changes in splice site usage. Asterisks indicated values deemed significant from control (shNT) at p<0.05.
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pone.0125315.g009: Tra2β Depletion Results in Selective Alterations in HIV-1 RNA Accumulation.HeLa rtTA HIVΔmls cells were transduced with lentiviruses expressing either control (shNT) or shRNA targeting Tra2β (shTra2β). After infection, transduced cells were selected using puromycin for 48–72 h, then HIV-1 provirus expression induced by addition of doxycycline. RNA was subsequently extracted and analyzed (A) by qRT-PCR to examine for changes in HIV-1 US, SS and MS RNA levels or (B) RT-PCR of MS RNAs to examine for changes in splice site usage. Asterisks indicated values deemed significant from control (shNT) at p<0.05.

Mentions: To complement the above overexpression studies and explore the role of the endogenous Tra2β in the regulation of HIV-1 RNA processing, targeted depletion studies were performed. To facilitate analysis, depletion experiments were performed in the context of the HeLa rtTA HIVΔmls cell line [37]. This cell line contains stably integrated, doxycycline (Dox) inducible HIV-1 provirus [46, 47]. which has a deletion of the reverse transcriptase and integrase reading frames to render it unable to replicate. Recent studies by our group have shown that overexpression of either Tra2β or Tra2βΔN in this cell line yielded similar alterations in HIV-1 RNA splice site selection as seen upon introduction into 293/293T cells used in this study [48]. To deplete Tra2β, cells were infected with lentivirus expressing control or shRNA to Tra2βand transduced cells isolated by treatment with puromycin. After puromycin selection for transduced cells, HIV-1 provirus expression was induced by addition of Dox for 24 h following which media and cells were harvested to analyze for changes in HIV-1 protein and RNA expression. As indicated in Fig 8A, significant depletion of Tra2β was achieved under the conditions used. Subsequent evaluation of HIV-1 Gag and Env protein levels revealed Tra2β depletion reduced Env synthesis slightly (~2 fold) (Fig 8B and 8C) but had a limited effect on Gag expression (Fig 8C). In light of these observations, we looked at whether these manipulations induced any changes in viral RNA abundance. qRTPCR analysis of HIV-1 RNA abundance determined that reduction of Tra2β levels resulted in a selective increase in MS RNA levels with no significant effect on US or SS RNA accumulation (Fig 9A). Parallel analysis of the MS RNA splicing pattern (Fig 9B) determined that there was no significant alteration in splice site use within this viral RNA class upon Tra2ß depletion.


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)

Tra2β Depletion Results in Selective Alterations in HIV-1 RNA Accumulation.HeLa rtTA HIVΔmls cells were transduced with lentiviruses expressing either control (shNT) or shRNA targeting Tra2β (shTra2β). After infection, transduced cells were selected using puromycin for 48–72 h, then HIV-1 provirus expression induced by addition of doxycycline. RNA was subsequently extracted and analyzed (A) by qRT-PCR to examine for changes in HIV-1 US, SS and MS RNA levels or (B) RT-PCR of MS RNAs to examine for changes in splice site usage. Asterisks indicated values deemed significant from control (shNT) at p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4430212&req=5

pone.0125315.g009: Tra2β Depletion Results in Selective Alterations in HIV-1 RNA Accumulation.HeLa rtTA HIVΔmls cells were transduced with lentiviruses expressing either control (shNT) or shRNA targeting Tra2β (shTra2β). After infection, transduced cells were selected using puromycin for 48–72 h, then HIV-1 provirus expression induced by addition of doxycycline. RNA was subsequently extracted and analyzed (A) by qRT-PCR to examine for changes in HIV-1 US, SS and MS RNA levels or (B) RT-PCR of MS RNAs to examine for changes in splice site usage. Asterisks indicated values deemed significant from control (shNT) at p<0.05.
Mentions: To complement the above overexpression studies and explore the role of the endogenous Tra2β in the regulation of HIV-1 RNA processing, targeted depletion studies were performed. To facilitate analysis, depletion experiments were performed in the context of the HeLa rtTA HIVΔmls cell line [37]. This cell line contains stably integrated, doxycycline (Dox) inducible HIV-1 provirus [46, 47]. which has a deletion of the reverse transcriptase and integrase reading frames to render it unable to replicate. Recent studies by our group have shown that overexpression of either Tra2β or Tra2βΔN in this cell line yielded similar alterations in HIV-1 RNA splice site selection as seen upon introduction into 293/293T cells used in this study [48]. To deplete Tra2β, cells were infected with lentivirus expressing control or shRNA to Tra2βand transduced cells isolated by treatment with puromycin. After puromycin selection for transduced cells, HIV-1 provirus expression was induced by addition of Dox for 24 h following which media and cells were harvested to analyze for changes in HIV-1 protein and RNA expression. As indicated in Fig 8A, significant depletion of Tra2β was achieved under the conditions used. Subsequent evaluation of HIV-1 Gag and Env protein levels revealed Tra2β depletion reduced Env synthesis slightly (~2 fold) (Fig 8B and 8C) but had a limited effect on Gag expression (Fig 8C). In light of these observations, we looked at whether these manipulations induced any changes in viral RNA abundance. qRTPCR analysis of HIV-1 RNA abundance determined that reduction of Tra2β levels resulted in a selective increase in MS RNA levels with no significant effect on US or SS RNA accumulation (Fig 9A). Parallel analysis of the MS RNA splicing pattern (Fig 9B) determined that there was no significant alteration in splice site use within this viral RNA class upon Tra2ß depletion.

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