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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

Quantification of Gag.L219A nuclear foci number with co-expression of SC35, SF2, or PSP1. (A) Representative images of QT6 cells co-transfected with Gag.L219A and SC35, SF2, or PSP1. (B) Quantification of the number of Gag.L219A nuclear foci in QT6 cells expressing Gag.L219A alone or Gag.L219A co-transfected with SC35, SF2, or PSP1. Mean and standard error of the mean are indicated for each transfection. Nuclear foci in at least 16 QT6 nuclei were counted. *denotes p = 0.0003.
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Figure 6: Quantification of Gag.L219A nuclear foci number with co-expression of SC35, SF2, or PSP1. (A) Representative images of QT6 cells co-transfected with Gag.L219A and SC35, SF2, or PSP1. (B) Quantification of the number of Gag.L219A nuclear foci in QT6 cells expressing Gag.L219A alone or Gag.L219A co-transfected with SC35, SF2, or PSP1. Mean and standard error of the mean are indicated for each transfection. Nuclear foci in at least 16 QT6 nuclei were counted. *denotes p = 0.0003.

Mentions: During the course of our imaging studies, we noticed that the number of nuclear Gag.L219A-CFP foci appeared to increase in cells that co-expressed SC35-YFP. To determine whether this effect was specific for SC35, we compared the number of nuclear Gag.L219A foci in cells expressing Gag.L219A-CFP alone compared to cells transfected with equal amounts of pSC35-YFP, pYFP-SF2, or pYFP-PSP1 (Figure 6A). In cells expressing Gag.L219A alone, the average number of Gag foci was approximately 22 per nucleus, whereas upon co-expression of SC35-YFP, the average number of Gag nucleoplasmic foci increased significantly to 36 (Figure 6B, p = 0.0003). By contrast, co-transfection of equal amounts of pGag.L219A with pYFP-SF2 or pYFP-PSP1 did not lead to a significant change in the number of nuclear Gag foci. This experiment was repeated in DF1 cells with the same outcome, indicating that the result was not specific to QT6 cells (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)

Quantification of Gag.L219A nuclear foci number with co-expression of SC35, SF2, or PSP1. (A) Representative images of QT6 cells co-transfected with Gag.L219A and SC35, SF2, or PSP1. (B) Quantification of the number of Gag.L219A nuclear foci in QT6 cells expressing Gag.L219A alone or Gag.L219A co-transfected with SC35, SF2, or PSP1. Mean and standard error of the mean are indicated for each transfection. Nuclear foci in at least 16 QT6 nuclei were counted. *denotes p = 0.0003.
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Related In: Results  -  Collection

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Figure 6: Quantification of Gag.L219A nuclear foci number with co-expression of SC35, SF2, or PSP1. (A) Representative images of QT6 cells co-transfected with Gag.L219A and SC35, SF2, or PSP1. (B) Quantification of the number of Gag.L219A nuclear foci in QT6 cells expressing Gag.L219A alone or Gag.L219A co-transfected with SC35, SF2, or PSP1. Mean and standard error of the mean are indicated for each transfection. Nuclear foci in at least 16 QT6 nuclei were counted. *denotes p = 0.0003.
Mentions: During the course of our imaging studies, we noticed that the number of nuclear Gag.L219A-CFP foci appeared to increase in cells that co-expressed SC35-YFP. To determine whether this effect was specific for SC35, we compared the number of nuclear Gag.L219A foci in cells expressing Gag.L219A-CFP alone compared to cells transfected with equal amounts of pSC35-YFP, pYFP-SF2, or pYFP-PSP1 (Figure 6A). In cells expressing Gag.L219A alone, the average number of Gag foci was approximately 22 per nucleus, whereas upon co-expression of SC35-YFP, the average number of Gag nucleoplasmic foci increased significantly to 36 (Figure 6B, p = 0.0003). By contrast, co-transfection of equal amounts of pGag.L219A with pYFP-SF2 or pYFP-PSP1 did not lead to a significant change in the number of nuclear Gag foci. This experiment was repeated in DF1 cells with the same outcome, indicating that the result was not specific to QT6 cells (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