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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

Characterization of Gag.L219A nuclear foci. (A) Schematics of the CFP-tagged wild-type RSV Gag protein (top) and the Gag.L219A nuclear export mutant (bottom), with leucine 219 in the p10 domain mutated to alanine. (B) Confocal micrographs of fluorescent protein-tagged wild-type Gag (left panel) and Gag.L219A (right panel) in QT6 cells. On the right hand image, the entire cell is outlined with a thin green line. (C) QT6 cells expressing Gag.L219A-CFP were imaged using time-lapse 3D confocal microscopy. A series of single optical slices through the nucleus were captured every 8 s for 10 min. After acquisition, the images were reconstructed as a 3D time-course using Imaris imaging software, and a single representative nucleus is shown with time = 0 on the left and t = 10 min on the right. The nucleus of each cell is outlined by a white dashed line. In the top panels, Gag foci (green) are shown. In the middle panels, the particle tracks were superimposed on the Gag foci, with white squares placed at the center of each focus (left) and tracks colored from blue (time = 0) to red (t = 10 min) in the middle panels. The particle tracks alone are shown in the bottom panel. In the lower left corner of the right image, a higher magnification of the particle tracks shows the course of the particles over the entire time period. (D) A histogram representing the anomalous diffusion coefficient α values for 149 nuclear foci is provided.
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Figure 1: Characterization of Gag.L219A nuclear foci. (A) Schematics of the CFP-tagged wild-type RSV Gag protein (top) and the Gag.L219A nuclear export mutant (bottom), with leucine 219 in the p10 domain mutated to alanine. (B) Confocal micrographs of fluorescent protein-tagged wild-type Gag (left panel) and Gag.L219A (right panel) in QT6 cells. On the right hand image, the entire cell is outlined with a thin green line. (C) QT6 cells expressing Gag.L219A-CFP were imaged using time-lapse 3D confocal microscopy. A series of single optical slices through the nucleus were captured every 8 s for 10 min. After acquisition, the images were reconstructed as a 3D time-course using Imaris imaging software, and a single representative nucleus is shown with time = 0 on the left and t = 10 min on the right. The nucleus of each cell is outlined by a white dashed line. In the top panels, Gag foci (green) are shown. In the middle panels, the particle tracks were superimposed on the Gag foci, with white squares placed at the center of each focus (left) and tracks colored from blue (time = 0) to red (t = 10 min) in the middle panels. The particle tracks alone are shown in the bottom panel. In the lower left corner of the right image, a higher magnification of the particle tracks shows the course of the particles over the entire time period. (D) A histogram representing the anomalous diffusion coefficient α values for 149 nuclear foci is provided.

Mentions: Treatment of RSV Gag expressing cells with the CRM1 inhibitor leptomycin B (LMB) traps Gag in the nucleus, and genetic mapping studies revealed a nuclear export signal (NES) in the p10 domain (Figure 1A). Mutation of hydrophobic residues within the NES causes Gag to accumulate in numerous, discrete nucleoplasmic foci and within nucleoli (Scheifele et al., 2002, 2005; Kenney et al., 2008; Lochmann et al., 2013). These nucleoplasmic foci are also observed at a lower frequency in the nuclei of cells expressing the wild-type Gag protein in the absence of LMB treatment (Figure 1B), providing evidence that formation of nuclear foci cannot be completely attributed to drug treatment or mutation. Furthermore, we demonstrated that Gag NES mutant proteins remain assembly-competent, as they interact with wild-type Gag proteins and can be rescued into virus particles (Kenney et al., 2008). The number and size of Gag nuclear foci increase with higher protein expression levels of the NES mutant Gag protein (data not shown), therefore it is possible that smaller accumulations of wild-type Gag proteins may form at lower expression levels, but these small foci are not readily detected by imaging studies.


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)

Characterization of Gag.L219A nuclear foci. (A) Schematics of the CFP-tagged wild-type RSV Gag protein (top) and the Gag.L219A nuclear export mutant (bottom), with leucine 219 in the p10 domain mutated to alanine. (B) Confocal micrographs of fluorescent protein-tagged wild-type Gag (left panel) and Gag.L219A (right panel) in QT6 cells. On the right hand image, the entire cell is outlined with a thin green line. (C) QT6 cells expressing Gag.L219A-CFP were imaged using time-lapse 3D confocal microscopy. A series of single optical slices through the nucleus were captured every 8 s for 10 min. After acquisition, the images were reconstructed as a 3D time-course using Imaris imaging software, and a single representative nucleus is shown with time = 0 on the left and t = 10 min on the right. The nucleus of each cell is outlined by a white dashed line. In the top panels, Gag foci (green) are shown. In the middle panels, the particle tracks were superimposed on the Gag foci, with white squares placed at the center of each focus (left) and tracks colored from blue (time = 0) to red (t = 10 min) in the middle panels. The particle tracks alone are shown in the bottom panel. In the lower left corner of the right image, a higher magnification of the particle tracks shows the course of the particles over the entire time period. (D) A histogram representing the anomalous diffusion coefficient α values for 149 nuclear foci is provided.
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Related In: Results  -  Collection

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Figure 1: Characterization of Gag.L219A nuclear foci. (A) Schematics of the CFP-tagged wild-type RSV Gag protein (top) and the Gag.L219A nuclear export mutant (bottom), with leucine 219 in the p10 domain mutated to alanine. (B) Confocal micrographs of fluorescent protein-tagged wild-type Gag (left panel) and Gag.L219A (right panel) in QT6 cells. On the right hand image, the entire cell is outlined with a thin green line. (C) QT6 cells expressing Gag.L219A-CFP were imaged using time-lapse 3D confocal microscopy. A series of single optical slices through the nucleus were captured every 8 s for 10 min. After acquisition, the images were reconstructed as a 3D time-course using Imaris imaging software, and a single representative nucleus is shown with time = 0 on the left and t = 10 min on the right. The nucleus of each cell is outlined by a white dashed line. In the top panels, Gag foci (green) are shown. In the middle panels, the particle tracks were superimposed on the Gag foci, with white squares placed at the center of each focus (left) and tracks colored from blue (time = 0) to red (t = 10 min) in the middle panels. The particle tracks alone are shown in the bottom panel. In the lower left corner of the right image, a higher magnification of the particle tracks shows the course of the particles over the entire time period. (D) A histogram representing the anomalous diffusion coefficient α values for 149 nuclear foci is provided.
Mentions: Treatment of RSV Gag expressing cells with the CRM1 inhibitor leptomycin B (LMB) traps Gag in the nucleus, and genetic mapping studies revealed a nuclear export signal (NES) in the p10 domain (Figure 1A). Mutation of hydrophobic residues within the NES causes Gag to accumulate in numerous, discrete nucleoplasmic foci and within nucleoli (Scheifele et al., 2002, 2005; Kenney et al., 2008; Lochmann et al., 2013). These nucleoplasmic foci are also observed at a lower frequency in the nuclei of cells expressing the wild-type Gag protein in the absence of LMB treatment (Figure 1B), providing evidence that formation of nuclear foci cannot be completely attributed to drug treatment or mutation. Furthermore, we demonstrated that Gag NES mutant proteins remain assembly-competent, as they interact with wild-type Gag proteins and can be rescued into virus particles (Kenney et al., 2008). The number and size of Gag nuclear foci increase with higher protein expression levels of the NES mutant Gag protein (data not shown), therefore it is possible that smaller accumulations of wild-type Gag proteins may form at lower expression levels, but these small foci are not readily detected by imaging studies.

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