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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

Localization of Gag.L219A with host nuclear body proteins in QT6 cells. (A) Localization of Gag.L219A and nuclear body proteins in singly transfected QT6 cells. (B) Co-localization analysis between Gag.L219A and the indicated nuclear body proteins in co-transfected QT6 cells. Merging of Gag.L219A and nuclear body marker protein channels is displayed (Overlay). The DAPI channel is also depicted. The percentage of Gag.L219A with each factor is depicted in the Gag.L219A channel with the standard error of the mean. (C) Scatterplot depicting the mean and standard error of the mean of Gag.L219A co-localization with each of the nuclear body protein. (D) Still image from Supplemental Video 1 that closely examines a surface rendering of SC35 (green) and Gag.L219A (red). (E) Still image from Supplemental Video 2 that closely examines a surface rendering of SF2 (green) and Gag.L219A (red).
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Figure 2: Localization of Gag.L219A with host nuclear body proteins in QT6 cells. (A) Localization of Gag.L219A and nuclear body proteins in singly transfected QT6 cells. (B) Co-localization analysis between Gag.L219A and the indicated nuclear body proteins in co-transfected QT6 cells. Merging of Gag.L219A and nuclear body marker protein channels is displayed (Overlay). The DAPI channel is also depicted. The percentage of Gag.L219A with each factor is depicted in the Gag.L219A channel with the standard error of the mean. (C) Scatterplot depicting the mean and standard error of the mean of Gag.L219A co-localization with each of the nuclear body protein. (D) Still image from Supplemental Video 1 that closely examines a surface rendering of SC35 (green) and Gag.L219A (red). (E) Still image from Supplemental Video 2 that closely examines a surface rendering of SF2 (green) and Gag.L219A (red).

Mentions: The particle tracking data suggest that Gag.L219A foci appeared to be tethered in foci which resemble subnuclear bodies. To determine whether Gag.L219A co-localized with host proteins in subnuclear bodies, we expressed fluorescently-tagged splicing factors that are components of speckles (SC35 and SF2); paraspeckles (PSP1, PSF, and p54nrb); or PML bodies (PML and SUMO1). Plasmids expressing these nuclear body components tagged with YFP were used to co-transfect avian (QT6) and human (HeLa) cells with pGag.L219A-CFP. We expected human SC35 and SF2 to form characteristic splicing speckles in QT6 cells due to the high level of conservation between human and chicken orthologs (98.19 and 98.79% amino acid identity, respectively). However, SC35 and SF2 appeared more diffuse in QT6 cells even when a low amount of plasmid DNA was used for transfection (100 ng), although there were areas of consolidation where the protein was concentrated (Figure 2A). Of interest, both SC35 and SF2 co-localized with Gag.L219A foci to a high degree. Quantitative Mander's analysis performed in 8 cells revealed that a mean of 69.8 ± 4.7% of Gag.L219A co-localized with SC35 and 61.5 ± 5.2% of Gag.L219A with SF2 (Figure 2C). To determine whether co-localization was present in 3-dimensions, z-stacks were obtained and reconstructions were performed using Imaris imaging analysis software (Supplemental Movie S2). Rendering of the Gag.L219A (red) and SC35 (green; Figure 2D, left) or SF2 (Figure 2D, right) signals revealed that Gag.L219A/SC35 and Gag.L219A/SF2 co-localized in the x, y, and z dimensions and appear to be in close proximity, at least based on the limits of resolution of the microscopic images obtained (theoretical resolution 250 nm in the x and y planes and 600 nm in the z plane). For Gag.L219A/SC35 and Gag.L219A/SF2, the Mander's co-localization values were statistically significantly higher (p < 0.0001 in both cases) than the quantitative co-localization measured between Gag.L219A and proteins that reside in paraspeckles (p54nrb, PSF, and PSP1) or PML bodies (PML and SUMO1) (Figures 2B,C). Together, these data suggest that Gag.L219A protein accumulated at subnuclear locations enriched in splicing speckle components 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)

Localization of Gag.L219A with host nuclear body proteins in QT6 cells. (A) Localization of Gag.L219A and nuclear body proteins in singly transfected QT6 cells. (B) Co-localization analysis between Gag.L219A and the indicated nuclear body proteins in co-transfected QT6 cells. Merging of Gag.L219A and nuclear body marker protein channels is displayed (Overlay). The DAPI channel is also depicted. The percentage of Gag.L219A with each factor is depicted in the Gag.L219A channel with the standard error of the mean. (C) Scatterplot depicting the mean and standard error of the mean of Gag.L219A co-localization with each of the nuclear body protein. (D) Still image from Supplemental Video 1 that closely examines a surface rendering of SC35 (green) and Gag.L219A (red). (E) Still image from Supplemental Video 2 that closely examines a surface rendering of SF2 (green) and Gag.L219A (red).
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Figure 2: Localization of Gag.L219A with host nuclear body proteins in QT6 cells. (A) Localization of Gag.L219A and nuclear body proteins in singly transfected QT6 cells. (B) Co-localization analysis between Gag.L219A and the indicated nuclear body proteins in co-transfected QT6 cells. Merging of Gag.L219A and nuclear body marker protein channels is displayed (Overlay). The DAPI channel is also depicted. The percentage of Gag.L219A with each factor is depicted in the Gag.L219A channel with the standard error of the mean. (C) Scatterplot depicting the mean and standard error of the mean of Gag.L219A co-localization with each of the nuclear body protein. (D) Still image from Supplemental Video 1 that closely examines a surface rendering of SC35 (green) and Gag.L219A (red). (E) Still image from Supplemental Video 2 that closely examines a surface rendering of SF2 (green) and Gag.L219A (red).
Mentions: The particle tracking data suggest that Gag.L219A foci appeared to be tethered in foci which resemble subnuclear bodies. To determine whether Gag.L219A co-localized with host proteins in subnuclear bodies, we expressed fluorescently-tagged splicing factors that are components of speckles (SC35 and SF2); paraspeckles (PSP1, PSF, and p54nrb); or PML bodies (PML and SUMO1). Plasmids expressing these nuclear body components tagged with YFP were used to co-transfect avian (QT6) and human (HeLa) cells with pGag.L219A-CFP. We expected human SC35 and SF2 to form characteristic splicing speckles in QT6 cells due to the high level of conservation between human and chicken orthologs (98.19 and 98.79% amino acid identity, respectively). However, SC35 and SF2 appeared more diffuse in QT6 cells even when a low amount of plasmid DNA was used for transfection (100 ng), although there were areas of consolidation where the protein was concentrated (Figure 2A). Of interest, both SC35 and SF2 co-localized with Gag.L219A foci to a high degree. Quantitative Mander's analysis performed in 8 cells revealed that a mean of 69.8 ± 4.7% of Gag.L219A co-localized with SC35 and 61.5 ± 5.2% of Gag.L219A with SF2 (Figure 2C). To determine whether co-localization was present in 3-dimensions, z-stacks were obtained and reconstructions were performed using Imaris imaging analysis software (Supplemental Movie S2). Rendering of the Gag.L219A (red) and SC35 (green; Figure 2D, left) or SF2 (Figure 2D, right) signals revealed that Gag.L219A/SC35 and Gag.L219A/SF2 co-localized in the x, y, and z dimensions and appear to be in close proximity, at least based on the limits of resolution of the microscopic images obtained (theoretical resolution 250 nm in the x and y planes and 600 nm in the z plane). For Gag.L219A/SC35 and Gag.L219A/SF2, the Mander's co-localization values were statistically significantly higher (p < 0.0001 in both cases) than the quantitative co-localization measured between Gag.L219A and proteins that reside in paraspeckles (p54nrb, PSF, and PSP1) or PML bodies (PML and SUMO1) (Figures 2B,C). Together, these data suggest that Gag.L219A protein accumulated at subnuclear locations enriched in splicing speckle components 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