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Interplay between the alpharetroviral Gag protein and SR proteins SF2 and SC35 in the nucleus.

Rice BL, Kaddis RJ, Stake MS, Lochmann TL, Parent LJ - Front Microbiol (2015)

Bottom Line: We previously reported that RSV Gag nuclear trafficking is required for efficient gRNA packaging.Together with the data presented herein, our findings raise the intriguing hypothesis that RSV Gag may co-opt splicing factors to localize near transcription sites.Because splicing occurs co-transcriptionally, we speculate that this mechanism could allow Gag to associate with unspliced viral RNA shortly after its transcription initiation in the nucleus, before the viral RNA can be spliced or exported from the nucleus as an mRNA template.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases and Epidemiology, Department of Medicine, Penn State College of Medicine Hershey, PA, USA.

ABSTRACT
Retroviruses are positive-sense, single-stranded RNA viruses that reverse transcribe their RNA genomes into double-stranded DNA for integration into the host cell chromosome. The integrated provirus is used as a template for the transcription of viral RNA. The full-length viral RNA can be used for the translation of the Gag and Gag-Pol structural proteins or as the genomic RNA (gRNA) for encapsidation into new virions by the Gag protein. The mechanism by which Gag selectively incorporates unspliced gRNA into virus particles is poorly understood. Although Gag was previously thought to localize exclusively to the cytoplasm and plasma membrane where particles are released, we found that the Gag protein of Rous sarcoma virus, an alpharetrovirus, undergoes transient nuclear trafficking. When the nuclear export signal of RSV Gag is mutated (Gag.L219A), the protein accumulates in discrete subnuclear foci reminiscent of nuclear bodies such as splicing speckles, paraspeckles, and PML bodies. In this report, we observed that RSV Gag.L219A foci appeared to be tethered in the nucleus, partially co-localizing with the splicing speckle components SC35 and SF2. Overexpression of SC35 increased the number of Gag.L219A nucleoplasmic foci, suggesting that SC35 may facilitate the formation of Gag foci. We previously reported that RSV Gag nuclear trafficking is required for efficient gRNA packaging. Together with the data presented herein, our findings raise the intriguing hypothesis that RSV Gag may co-opt splicing factors to localize near transcription sites. Because splicing occurs co-transcriptionally, we speculate that this mechanism could allow Gag to associate with unspliced viral RNA shortly after its transcription initiation in the nucleus, before the viral RNA can be spliced or exported from the nucleus as an mRNA template.

No MeSH data available.


Related in: MedlinePlus

The effect of Clk1 on Gag.L219A localization in QT6 cells. (A) Localization of Gag.L219A, Clk1, SC35, or SF2 in singly transfected QT6 cells. (B) Co-expression of Clk1 with Gag.L219A, SC35, or SF2 in QT6 cells. “Overlay” depicts a merge of the Clk1 and Gag.L219A or splicing factor channels. The DAPI channel is also displayed. The percentage of Gag.L219A, SC35, or SF2 co-localizing with Clk1 is depicted in the corresponding Gag.L219A, SC35, and SF2 channels with the standard error of the mean. (C) QT6 cells co-transfected with Gag.L219A, Clk1, and SC35 (top panel) or SF2 (bottom panel). Merging the Gag.L219A and splicing factor channels results in the “Overlay” channel. The DAPI channel is also displayed. The percentage of Gag.L219A co-localization with SC35 or SF2 in the presence of Clk1 overexpression is depicted in the corresponding Gag.L219A channel with the standard error of the mean.
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Figure 5: The effect of Clk1 on Gag.L219A localization in QT6 cells. (A) Localization of Gag.L219A, Clk1, SC35, or SF2 in singly transfected QT6 cells. (B) Co-expression of Clk1 with Gag.L219A, SC35, or SF2 in QT6 cells. “Overlay” depicts a merge of the Clk1 and Gag.L219A or splicing factor channels. The DAPI channel is also displayed. The percentage of Gag.L219A, SC35, or SF2 co-localizing with Clk1 is depicted in the corresponding Gag.L219A, SC35, and SF2 channels with the standard error of the mean. (C) QT6 cells co-transfected with Gag.L219A, Clk1, and SC35 (top panel) or SF2 (bottom panel). Merging the Gag.L219A and splicing factor channels results in the “Overlay” channel. The DAPI channel is also displayed. The percentage of Gag.L219A co-localization with SC35 or SF2 in the presence of Clk1 overexpression is depicted in the corresponding Gag.L219A channel with the standard error of the mean.

Mentions: Phosphorylation is a major mechanism for regulating the localization of SR proteins in the nucleus (Yeakley et al., 1999), therefore we examined whether the degree of phosphorylation of splicing factors SC35 and SF2 influenced their association with Gag.L219A. To that end, murine Clk1, an SR protein kinase (SRPK) that phosphorylates the RS domains of SC35 and SF2 (Gui et al., 1994; Colwill et al., 1996; Nayler et al., 1997; Koizumi et al., 1999; Aubol et al., 2002; Ngo et al., 2005) was expressed as an mCherry fusion protein in QT6 cells, either alone (Figure 5A) or in conjunction with Gag.L219A-CFP, SC35-YFP or YFP-SF2 (Figure 5B). When co-expressed with Gag.L219A, there was no significant co-localization with Clk1-mCherry (Figure 5B, upper row). However, as expected, there was co-localization between Clk1/SC35 and Clk1/SF2 (Figure 5B, lower rows) because Clk1 phosphorylates both SC35 and SF2.


Interplay between the alpharetroviral Gag protein and SR proteins SF2 and SC35 in the nucleus.

Rice BL, Kaddis RJ, Stake MS, Lochmann TL, Parent LJ - Front Microbiol (2015)

The effect of Clk1 on Gag.L219A localization in QT6 cells. (A) Localization of Gag.L219A, Clk1, SC35, or SF2 in singly transfected QT6 cells. (B) Co-expression of Clk1 with Gag.L219A, SC35, or SF2 in QT6 cells. “Overlay” depicts a merge of the Clk1 and Gag.L219A or splicing factor channels. The DAPI channel is also displayed. The percentage of Gag.L219A, SC35, or SF2 co-localizing with Clk1 is depicted in the corresponding Gag.L219A, SC35, and SF2 channels with the standard error of the mean. (C) QT6 cells co-transfected with Gag.L219A, Clk1, and SC35 (top panel) or SF2 (bottom panel). Merging the Gag.L219A and splicing factor channels results in the “Overlay” channel. The DAPI channel is also displayed. The percentage of Gag.L219A co-localization with SC35 or SF2 in the presence of Clk1 overexpression is depicted in the corresponding Gag.L219A channel with the standard error of the mean.
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Figure 5: The effect of Clk1 on Gag.L219A localization in QT6 cells. (A) Localization of Gag.L219A, Clk1, SC35, or SF2 in singly transfected QT6 cells. (B) Co-expression of Clk1 with Gag.L219A, SC35, or SF2 in QT6 cells. “Overlay” depicts a merge of the Clk1 and Gag.L219A or splicing factor channels. The DAPI channel is also displayed. The percentage of Gag.L219A, SC35, or SF2 co-localizing with Clk1 is depicted in the corresponding Gag.L219A, SC35, and SF2 channels with the standard error of the mean. (C) QT6 cells co-transfected with Gag.L219A, Clk1, and SC35 (top panel) or SF2 (bottom panel). Merging the Gag.L219A and splicing factor channels results in the “Overlay” channel. The DAPI channel is also displayed. The percentage of Gag.L219A co-localization with SC35 or SF2 in the presence of Clk1 overexpression is depicted in the corresponding Gag.L219A channel with the standard error of the mean.
Mentions: Phosphorylation is a major mechanism for regulating the localization of SR proteins in the nucleus (Yeakley et al., 1999), therefore we examined whether the degree of phosphorylation of splicing factors SC35 and SF2 influenced their association with Gag.L219A. To that end, murine Clk1, an SR protein kinase (SRPK) that phosphorylates the RS domains of SC35 and SF2 (Gui et al., 1994; Colwill et al., 1996; Nayler et al., 1997; Koizumi et al., 1999; Aubol et al., 2002; Ngo et al., 2005) was expressed as an mCherry fusion protein in QT6 cells, either alone (Figure 5A) or in conjunction with Gag.L219A-CFP, SC35-YFP or YFP-SF2 (Figure 5B). When co-expressed with Gag.L219A, there was no significant co-localization with Clk1-mCherry (Figure 5B, upper row). However, as expected, there was co-localization between Clk1/SC35 and Clk1/SF2 (Figure 5B, lower rows) because Clk1 phosphorylates both SC35 and SF2.

Bottom Line: We previously reported that RSV Gag nuclear trafficking is required for efficient gRNA packaging.Together with the data presented herein, our findings raise the intriguing hypothesis that RSV Gag may co-opt splicing factors to localize near transcription sites.Because splicing occurs co-transcriptionally, we speculate that this mechanism could allow Gag to associate with unspliced viral RNA shortly after its transcription initiation in the nucleus, before the viral RNA can be spliced or exported from the nucleus as an mRNA template.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases and Epidemiology, Department of Medicine, Penn State College of Medicine Hershey, PA, USA.

ABSTRACT
Retroviruses are positive-sense, single-stranded RNA viruses that reverse transcribe their RNA genomes into double-stranded DNA for integration into the host cell chromosome. The integrated provirus is used as a template for the transcription of viral RNA. The full-length viral RNA can be used for the translation of the Gag and Gag-Pol structural proteins or as the genomic RNA (gRNA) for encapsidation into new virions by the Gag protein. The mechanism by which Gag selectively incorporates unspliced gRNA into virus particles is poorly understood. Although Gag was previously thought to localize exclusively to the cytoplasm and plasma membrane where particles are released, we found that the Gag protein of Rous sarcoma virus, an alpharetrovirus, undergoes transient nuclear trafficking. When the nuclear export signal of RSV Gag is mutated (Gag.L219A), the protein accumulates in discrete subnuclear foci reminiscent of nuclear bodies such as splicing speckles, paraspeckles, and PML bodies. In this report, we observed that RSV Gag.L219A foci appeared to be tethered in the nucleus, partially co-localizing with the splicing speckle components SC35 and SF2. Overexpression of SC35 increased the number of Gag.L219A nucleoplasmic foci, suggesting that SC35 may facilitate the formation of Gag foci. We previously reported that RSV Gag nuclear trafficking is required for efficient gRNA packaging. Together with the data presented herein, our findings raise the intriguing hypothesis that RSV Gag may co-opt splicing factors to localize near transcription sites. Because splicing occurs co-transcriptionally, we speculate that this mechanism could allow Gag to associate with unspliced viral RNA shortly after its transcription initiation in the nucleus, before the viral RNA can be spliced or exported from the nucleus as an mRNA template.

No MeSH data available.


Related in: MedlinePlus