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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 HeLa cells. (A) Localization of Gag.L219A and nuclear body proteins in singly transfected HeLa cells (B) Co-localization analysis between Gag.L219A and the indicated nuclear body proteins in co-transfected HeLa 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 proteins.
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Figure 3: Localization of Gag.L219A with host nuclear body proteins in HeLa cells. (A) Localization of Gag.L219A and nuclear body proteins in singly transfected HeLa cells (B) Co-localization analysis between Gag.L219A and the indicated nuclear body proteins in co-transfected HeLa 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 proteins.

Mentions: Next, because we observed co-localization between Gag.L219A and human SC35 and SF2 expressed in avian cells, we wanted to determine whether similar co-localization patterns would be observed in human cells, in which the localization of splicing factors has been more extensively studied. When expressed alone, SC35 adopted its characteristic speckled appearance; however, co-expression with Gag.L219A resulted in a more diffuse pattern, and there was a high degree of co-localization between the proteins (79.7 ± 2.5% of Gag co-localized with SC35; Figures 3A,B). SF2 accumulated in speckles when expressed alone, and SF2 also showed a high degree of co-localization with Gag.L219A (60.6 ± 6%). Gag co-localization was significantly higher with SC35 compared to SF2 (p = 0.0192) and both SC35 and SF2 were more strongly associated with Gag.L219A compared to p54nrb, PSF or PSP1 (p < 0.0001). Analysis of cells co-expressing Gag.L219A and PML or SUMO1 could not be performed due to cell toxicity (data not shown).


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 HeLa cells. (A) Localization of Gag.L219A and nuclear body proteins in singly transfected HeLa cells (B) Co-localization analysis between Gag.L219A and the indicated nuclear body proteins in co-transfected HeLa 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 proteins.
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Related In: Results  -  Collection

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Figure 3: Localization of Gag.L219A with host nuclear body proteins in HeLa cells. (A) Localization of Gag.L219A and nuclear body proteins in singly transfected HeLa cells (B) Co-localization analysis between Gag.L219A and the indicated nuclear body proteins in co-transfected HeLa 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 proteins.
Mentions: Next, because we observed co-localization between Gag.L219A and human SC35 and SF2 expressed in avian cells, we wanted to determine whether similar co-localization patterns would be observed in human cells, in which the localization of splicing factors has been more extensively studied. When expressed alone, SC35 adopted its characteristic speckled appearance; however, co-expression with Gag.L219A resulted in a more diffuse pattern, and there was a high degree of co-localization between the proteins (79.7 ± 2.5% of Gag co-localized with SC35; Figures 3A,B). SF2 accumulated in speckles when expressed alone, and SF2 also showed a high degree of co-localization with Gag.L219A (60.6 ± 6%). Gag co-localization was significantly higher with SC35 compared to SF2 (p = 0.0192) and both SC35 and SF2 were more strongly associated with Gag.L219A compared to p54nrb, PSF or PSP1 (p < 0.0001). Analysis of cells co-expressing Gag.L219A and PML or SUMO1 could not be performed due to cell toxicity (data not shown).

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