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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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Related in: MedlinePlus

Preventing Phospho-serine 2 RNAP II Degradation Also Prevents Hsc70 Focus Formation.Vero cells that were mock-infected or were infected with WT HSV-1 or 27-LacZ were untreated (left panels) or were treated with 50 µM MG132 added 1 h after infection (right panels). Cells were fixed at 8 h after infection and stained with anti-Hsc70 antibody, antibody H5 and anti-ICP4 antibody as indicated. Arrows mark Hsc70 foci (green) or replication or pre-replication sites (red).
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pone-0001491-g008: Preventing Phospho-serine 2 RNAP II Degradation Also Prevents Hsc70 Focus Formation.Vero cells that were mock-infected or were infected with WT HSV-1 or 27-LacZ were untreated (left panels) or were treated with 50 µM MG132 added 1 h after infection (right panels). Cells were fixed at 8 h after infection and stained with anti-Hsc70 antibody, antibody H5 and anti-ICP4 antibody as indicated. Arrows mark Hsc70 foci (green) or replication or pre-replication sites (red).

Mentions: The degradation of RNAP II during HSV-1 infection can be prevented by addition of the proteasomal inhibitor MG132 [11]. We asked what effect MG132 would have on Hsc70 focus formation. There was no change in the diffuse nuclear and cytoplasmic staining of Hsc70 in mock and 27-LacZ-infected cells with or without MG132 (Figure 8). In contrast, Hsc70 nuclear focus formation was curtailed in WT HSV-1-infected cells in the presence of MG132, as was the appearance of H5 speckled structures (Figure 8). Therefore, preventing the proteasomal degradation of RNAP II during HSV-1 infection also precludes the formation of Hsc70 nuclear foci.


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)

Preventing Phospho-serine 2 RNAP II Degradation Also Prevents Hsc70 Focus Formation.Vero cells that were mock-infected or were infected with WT HSV-1 or 27-LacZ were untreated (left panels) or were treated with 50 µM MG132 added 1 h after infection (right panels). Cells were fixed at 8 h after infection and stained with anti-Hsc70 antibody, antibody H5 and anti-ICP4 antibody as indicated. Arrows mark Hsc70 foci (green) or replication or pre-replication sites (red).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2200795&req=5

pone-0001491-g008: Preventing Phospho-serine 2 RNAP II Degradation Also Prevents Hsc70 Focus Formation.Vero cells that were mock-infected or were infected with WT HSV-1 or 27-LacZ were untreated (left panels) or were treated with 50 µM MG132 added 1 h after infection (right panels). Cells were fixed at 8 h after infection and stained with anti-Hsc70 antibody, antibody H5 and anti-ICP4 antibody as indicated. Arrows mark Hsc70 foci (green) or replication or pre-replication sites (red).
Mentions: The degradation of RNAP II during HSV-1 infection can be prevented by addition of the proteasomal inhibitor MG132 [11]. We asked what effect MG132 would have on Hsc70 focus formation. There was no change in the diffuse nuclear and cytoplasmic staining of Hsc70 in mock and 27-LacZ-infected cells with or without MG132 (Figure 8). In contrast, Hsc70 nuclear focus formation was curtailed in WT HSV-1-infected cells in the presence of MG132, as was the appearance of H5 speckled structures (Figure 8). Therefore, preventing the proteasomal degradation of RNAP II during HSV-1 infection also precludes the formation of Hsc70 nuclear foci.

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