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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

Gag.L219A localization with endogenous splicing speckles in HeLa cells. (A) Visualization of endogenous splicing speckles in HeLa cells using immunofluorescence with α-phospho RS antibody (also called α-SC35 antibody; see Materials and Methods). (B) Localization of Gag.L219A in unstained HeLa cells fixed and permeabilized using the conditions outlined in Materials and Methods used to visualize endogenous splicing speckles. (C) Gag.L219A transfected HeLa cells were stained with α-phospho RS antibody. The Gag.L219A and α-phospho RS channels were combined (Overlay). This merged image was overlaid with the DAPI channel (Overlay with DAPI). The same region from each channel denoted by the white box was cropped, enlarged, and displayed in the bottom panel. The intensity of α-phospho RS antibody decreased in the presence of Gag.L219A (indicated by solid arrowhead) compared to cells in which Gag.L219A was not expressed (indicated by open arrowhead) (p < 0.001; 6 cells were analyzed per condition).
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Figure 4: Gag.L219A localization with endogenous splicing speckles in HeLa cells. (A) Visualization of endogenous splicing speckles in HeLa cells using immunofluorescence with α-phospho RS antibody (also called α-SC35 antibody; see Materials and Methods). (B) Localization of Gag.L219A in unstained HeLa cells fixed and permeabilized using the conditions outlined in Materials and Methods used to visualize endogenous splicing speckles. (C) Gag.L219A transfected HeLa cells were stained with α-phospho RS antibody. The Gag.L219A and α-phospho RS channels were combined (Overlay). This merged image was overlaid with the DAPI channel (Overlay with DAPI). The same region from each channel denoted by the white box was cropped, enlarged, and displayed in the bottom panel. The intensity of α-phospho RS antibody decreased in the presence of Gag.L219A (indicated by solid arrowhead) compared to cells in which Gag.L219A was not expressed (indicated by open arrowhead) (p < 0.001; 6 cells were analyzed per condition).

Mentions: To determine whether Gag.L219A foci co-localized with endogenous nuclear splicing speckles, we performed immunofluorescence using an antibody that recognizes the phosphorylated RS domains of SF2 and SC35 (Figure 4A). Gag.L219A accumulated in nuclear foci in transfected HeLa cells that appeared similar to those observed in QT6 cells (Figure 4B). In cells expressing Gag.L219A, overlapping signals were observed at the intersection of the phosphorylated SR domain proteins and Gag.L219A foci, which appeared to be juxtaposed (see enlarged image in bottom row of Figure 4C), suggesting that Gag foci form near accumulations of splicing speckle components. Of note, in HeLa cells expressing Gag.L219A, the amount of endogenous SC35/SF2 staining was dramatically reduced, as indicated by the statistically significant (p < 0.001) decrease in the mean signal intensity of the anti-phospho RS antibody channel (Figure 4C, solid arrowhead) compared to cells in which there was no Gag expression (open arrowhead). This result suggests that expression of Gag.L219A interferes with staining of endogenous phospho RS domains of splicing factors, although the mechanism remains unclear.


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)

Gag.L219A localization with endogenous splicing speckles in HeLa cells. (A) Visualization of endogenous splicing speckles in HeLa cells using immunofluorescence with α-phospho RS antibody (also called α-SC35 antibody; see Materials and Methods). (B) Localization of Gag.L219A in unstained HeLa cells fixed and permeabilized using the conditions outlined in Materials and Methods used to visualize endogenous splicing speckles. (C) Gag.L219A transfected HeLa cells were stained with α-phospho RS antibody. The Gag.L219A and α-phospho RS channels were combined (Overlay). This merged image was overlaid with the DAPI channel (Overlay with DAPI). The same region from each channel denoted by the white box was cropped, enlarged, and displayed in the bottom panel. The intensity of α-phospho RS antibody decreased in the presence of Gag.L219A (indicated by solid arrowhead) compared to cells in which Gag.L219A was not expressed (indicated by open arrowhead) (p < 0.001; 6 cells were analyzed per condition).
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Figure 4: Gag.L219A localization with endogenous splicing speckles in HeLa cells. (A) Visualization of endogenous splicing speckles in HeLa cells using immunofluorescence with α-phospho RS antibody (also called α-SC35 antibody; see Materials and Methods). (B) Localization of Gag.L219A in unstained HeLa cells fixed and permeabilized using the conditions outlined in Materials and Methods used to visualize endogenous splicing speckles. (C) Gag.L219A transfected HeLa cells were stained with α-phospho RS antibody. The Gag.L219A and α-phospho RS channels were combined (Overlay). This merged image was overlaid with the DAPI channel (Overlay with DAPI). The same region from each channel denoted by the white box was cropped, enlarged, and displayed in the bottom panel. The intensity of α-phospho RS antibody decreased in the presence of Gag.L219A (indicated by solid arrowhead) compared to cells in which Gag.L219A was not expressed (indicated by open arrowhead) (p < 0.001; 6 cells were analyzed per condition).
Mentions: To determine whether Gag.L219A foci co-localized with endogenous nuclear splicing speckles, we performed immunofluorescence using an antibody that recognizes the phosphorylated RS domains of SF2 and SC35 (Figure 4A). Gag.L219A accumulated in nuclear foci in transfected HeLa cells that appeared similar to those observed in QT6 cells (Figure 4B). In cells expressing Gag.L219A, overlapping signals were observed at the intersection of the phosphorylated SR domain proteins and Gag.L219A foci, which appeared to be juxtaposed (see enlarged image in bottom row of Figure 4C), suggesting that Gag foci form near accumulations of splicing speckle components. Of note, in HeLa cells expressing Gag.L219A, the amount of endogenous SC35/SF2 staining was dramatically reduced, as indicated by the statistically significant (p < 0.001) decrease in the mean signal intensity of the anti-phospho RS antibody channel (Figure 4C, solid arrowhead) compared to cells in which there was no Gag expression (open arrowhead). This result suggests that expression of Gag.L219A interferes with staining of endogenous phospho RS domains of splicing factors, although the mechanism remains unclear.

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