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Hsc70 focus formation at the periphery of HSV-1 transcription sites requires ICP27.

Li L, Johnson LA, Dai-Ju JQ, Sandri-Goldin RM - PLoS ONE (2008)

Bottom Line: During infection with ICP27 mutants that are unable to recruit RNAP II to viral replication sites, viral transcript levels were greatly reduced, viral replication compartments were poorly formed and Hsc70 focus formation was curtailed.Further, a dominant negative Hsc70 mutant that cannot hydrolyze ATP, interfered with RNAP II degradation during HSV-1 infection, and an increase in ubiquitinated forms of RNAP II was observed.We propose that one function of the Hsc70 nuclear foci may be to serve to facilitate the process of clearing stalled RNAP II complexes from viral genomes during times of highly active transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, California, USA.

ABSTRACT

Background: The cellular chaperone protein Hsc70, along with components of the 26S proteasome and ubiquitin-conjugated proteins have been shown to be sequestered in discrete foci in the nuclei of herpes simplex virus 1 (HSV-1) infected cells. We recently reported that cellular RNA polymerase II (RNAP II) undergoes proteasomal degradation during robust HSV-1 transcription, and that the immediate early protein ICP27 interacts with the C-terminal domain and is involved in the recruitment of RNAP II to viral transcription/replication compartments.

Methodology/principle findings: Here we show that ICP27 also interacts with Hsc70, and is required for the formation of Hsc70 nuclear foci. During infection with ICP27 mutants that are unable to recruit RNAP II to viral replication sites, viral transcript levels were greatly reduced, viral replication compartments were poorly formed and Hsc70 focus formation was curtailed. Further, a dominant negative Hsc70 mutant that cannot hydrolyze ATP, interfered with RNAP II degradation during HSV-1 infection, and an increase in ubiquitinated forms of RNAP II was observed. There was also a decrease in virus yields, indicating that proteasomal degradation of stalled RNAP II complexes during robust HSV-1 transcription and replication benefits viral gene expression.

Conclusions/significance: We propose that one function of the Hsc70 nuclear foci may be to serve to facilitate the process of clearing stalled RNAP II complexes from viral genomes during times of highly active transcription.

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Hsc70 Nuclear Focus Formation Is Delayed in Infections with ICP27 Mutants.A) Vero cells were infected with WT HSV-1 or mutants dLeu, n406 or n504. At the times indicated, cells were fixed and stained with anti-Hsc70 and anti-ICP27 antibodies as indicated. Arrows mark Hsc70 nuclear foci. B) Vero cells were infected with WT HSV-1, 27-LacZ, dLeu, n406 or n504 for 6 h, at which time cells were fixed and stained with anti-Hsc70 and anti-ICP0 antibodies. The arrow marks an Hsc70 focus adjacent to an ICP0 speckle.
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pone-0001491-g004: Hsc70 Nuclear Focus Formation Is Delayed in Infections with ICP27 Mutants.A) Vero cells were infected with WT HSV-1 or mutants dLeu, n406 or n504. At the times indicated, cells were fixed and stained with anti-Hsc70 and anti-ICP27 antibodies as indicated. Arrows mark Hsc70 nuclear foci. B) Vero cells were infected with WT HSV-1, 27-LacZ, dLeu, n406 or n504 for 6 h, at which time cells were fixed and stained with anti-Hsc70 and anti-ICP0 antibodies. The arrow marks an Hsc70 focus adjacent to an ICP0 speckle.

Mentions: To determine if Hsc70 was relocalized to nuclear foci during infection with ICP27 mutants that do not interact with Hsc70, immunofluorescent staining was performed. In cells infected with WT HSV-1, Hsc70 focus formation was clearly seen by 6 h after infection, a time when ICP27 is still strongly nuclear, but it has begun to shuttle to the cytoplasm in its role as a viral RNA export factor (Figure 4A). The shuttling of ICP27 can be seen more clearly at 8 h after infection (Figure 4A). In contrast, in dLeu-infected cells, Hsc70 foci were not observed even by 8 h after infection; while in n406-infected cells, small Hsc70 foci were seen but not until 8 h after infection, when larger foci are apparent in WT-infected cells (Figure 4A). Because viral replication is both curtailed and delayed in n406 infected cells, the smaller foci seen at 8 h in n406 infections, probably reflect the timing of the beginning of DNA replication. Further, both dLeu and n406 are confined to the nucleus because these ICP27 mutants do not interact with TAP/NXF, which is required for ICP27 to shuttle to the cytoplasm [29]. Interestingly, mutant n504, which also does not interact with TAP/NXF1 was confined to the nucleus at 6 h after infection, however, Hsc70 foci were seen to form. This mutant does interact with Hsc70.


Hsc70 focus formation at the periphery of HSV-1 transcription sites requires ICP27.

Li L, Johnson LA, Dai-Ju JQ, Sandri-Goldin RM - PLoS ONE (2008)

Hsc70 Nuclear Focus Formation Is Delayed in Infections with ICP27 Mutants.A) Vero cells were infected with WT HSV-1 or mutants dLeu, n406 or n504. At the times indicated, cells were fixed and stained with anti-Hsc70 and anti-ICP27 antibodies as indicated. Arrows mark Hsc70 nuclear foci. B) Vero cells were infected with WT HSV-1, 27-LacZ, dLeu, n406 or n504 for 6 h, at which time cells were fixed and stained with anti-Hsc70 and anti-ICP0 antibodies. The arrow marks an Hsc70 focus adjacent to an ICP0 speckle.
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Related In: Results  -  Collection

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pone-0001491-g004: Hsc70 Nuclear Focus Formation Is Delayed in Infections with ICP27 Mutants.A) Vero cells were infected with WT HSV-1 or mutants dLeu, n406 or n504. At the times indicated, cells were fixed and stained with anti-Hsc70 and anti-ICP27 antibodies as indicated. Arrows mark Hsc70 nuclear foci. B) Vero cells were infected with WT HSV-1, 27-LacZ, dLeu, n406 or n504 for 6 h, at which time cells were fixed and stained with anti-Hsc70 and anti-ICP0 antibodies. The arrow marks an Hsc70 focus adjacent to an ICP0 speckle.
Mentions: To determine if Hsc70 was relocalized to nuclear foci during infection with ICP27 mutants that do not interact with Hsc70, immunofluorescent staining was performed. In cells infected with WT HSV-1, Hsc70 focus formation was clearly seen by 6 h after infection, a time when ICP27 is still strongly nuclear, but it has begun to shuttle to the cytoplasm in its role as a viral RNA export factor (Figure 4A). The shuttling of ICP27 can be seen more clearly at 8 h after infection (Figure 4A). In contrast, in dLeu-infected cells, Hsc70 foci were not observed even by 8 h after infection; while in n406-infected cells, small Hsc70 foci were seen but not until 8 h after infection, when larger foci are apparent in WT-infected cells (Figure 4A). Because viral replication is both curtailed and delayed in n406 infected cells, the smaller foci seen at 8 h in n406 infections, probably reflect the timing of the beginning of DNA replication. Further, both dLeu and n406 are confined to the nucleus because these ICP27 mutants do not interact with TAP/NXF, which is required for ICP27 to shuttle to the cytoplasm [29]. Interestingly, mutant n504, which also does not interact with TAP/NXF1 was confined to the nucleus at 6 h after infection, however, Hsc70 foci were seen to form. This mutant does interact with Hsc70.

Bottom Line: During infection with ICP27 mutants that are unable to recruit RNAP II to viral replication sites, viral transcript levels were greatly reduced, viral replication compartments were poorly formed and Hsc70 focus formation was curtailed.Further, a dominant negative Hsc70 mutant that cannot hydrolyze ATP, interfered with RNAP II degradation during HSV-1 infection, and an increase in ubiquitinated forms of RNAP II was observed.We propose that one function of the Hsc70 nuclear foci may be to serve to facilitate the process of clearing stalled RNAP II complexes from viral genomes during times of highly active transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, California, USA.

ABSTRACT

Background: The cellular chaperone protein Hsc70, along with components of the 26S proteasome and ubiquitin-conjugated proteins have been shown to be sequestered in discrete foci in the nuclei of herpes simplex virus 1 (HSV-1) infected cells. We recently reported that cellular RNA polymerase II (RNAP II) undergoes proteasomal degradation during robust HSV-1 transcription, and that the immediate early protein ICP27 interacts with the C-terminal domain and is involved in the recruitment of RNAP II to viral transcription/replication compartments.

Methodology/principle findings: Here we show that ICP27 also interacts with Hsc70, and is required for the formation of Hsc70 nuclear foci. During infection with ICP27 mutants that are unable to recruit RNAP II to viral replication sites, viral transcript levels were greatly reduced, viral replication compartments were poorly formed and Hsc70 focus formation was curtailed. Further, a dominant negative Hsc70 mutant that cannot hydrolyze ATP, interfered with RNAP II degradation during HSV-1 infection, and an increase in ubiquitinated forms of RNAP II was observed. There was also a decrease in virus yields, indicating that proteasomal degradation of stalled RNAP II complexes during robust HSV-1 transcription and replication benefits viral gene expression.

Conclusions/significance: We propose that one function of the Hsc70 nuclear foci may be to serve to facilitate the process of clearing stalled RNAP II complexes from viral genomes during times of highly active transcription.

Show MeSH
Related in: MedlinePlus