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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 Sequestration Correlates with the Loss of Phospho-serine 2 Form of RNAP II CTD in Fully Formed Replication Compartments.Vero cells were mock-infected or infected with WT HSV-1, dLeu or n406 for the times indicated. In the left hand panels, cells were stained with anti-RNAP II antibody, H14, which recognizes phospho-serine 5 RNAP II CTD, and anti-Hsc70 antibody. Arrows show a replication compartment with an Hsc70 focus site at the periphery in the 8 h WT-infected cell, and a pre-replication site (red) next to a small Hsc70 focus site (merge) in the 12 h dLeu-infected cell. In the right panels, cells were stained with anti-Hsc70 antibody and anti-RNAP II antibody H5, which recognizes phospho-serine-2 RNAP II CTD. This antibody also cross-reacts with a phospho-epitope in splicing SR proteins [11], [40]. The arrow points to an Hsc70 focus site (green) adjacent to a splicing speckle (red).
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pone-0001491-g007: Hsc70 Nuclear Sequestration Correlates with the Loss of Phospho-serine 2 Form of RNAP II CTD in Fully Formed Replication Compartments.Vero cells were mock-infected or infected with WT HSV-1, dLeu or n406 for the times indicated. In the left hand panels, cells were stained with anti-RNAP II antibody, H14, which recognizes phospho-serine 5 RNAP II CTD, and anti-Hsc70 antibody. Arrows show a replication compartment with an Hsc70 focus site at the periphery in the 8 h WT-infected cell, and a pre-replication site (red) next to a small Hsc70 focus site (merge) in the 12 h dLeu-infected cell. In the right panels, cells were stained with anti-Hsc70 antibody and anti-RNAP II antibody H5, which recognizes phospho-serine-2 RNAP II CTD. This antibody also cross-reacts with a phospho-epitope in splicing SR proteins [11], [40]. The arrow points to an Hsc70 focus site (green) adjacent to a splicing speckle (red).

Mentions: To monitor relocalization of RNAP II to viral replication compartments, staining was performed with monoclonal antibody H14, which recognizes the serine-5 phosphorylated form of RNAP II CTD found in the initiation complex [38], [39]. Serine-5 phosphorylated RNAP II was recruited to structures resembling viral replication compartments by 8 h after infection in WT HSV-1-infected cells, and Hsc70 foci formed at the periphery of these structures (Figure 7, left panels). To show that these structures were indeed replication compartments, H14 stained cells were also stained with antibody to ICP4 (Figure S1). It is clear that serine-5 RNAP II stained by H14 antibody colocalized with ICP4-marked replication compartments. In contrast, in dLeu and n406 infections, H14 staining was similar to mock infected cells at 8 h for dLeu and 6 h for n406-infected cells (Figure 7, left panels). It was not until later times with these mutants that small Hsc70 foci were seen to form along with small compartments that stained with H14, and which presumably represent viral pre-replication sites (Figure 7, left panels and Figure S1).


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 Sequestration Correlates with the Loss of Phospho-serine 2 Form of RNAP II CTD in Fully Formed Replication Compartments.Vero cells were mock-infected or infected with WT HSV-1, dLeu or n406 for the times indicated. In the left hand panels, cells were stained with anti-RNAP II antibody, H14, which recognizes phospho-serine 5 RNAP II CTD, and anti-Hsc70 antibody. Arrows show a replication compartment with an Hsc70 focus site at the periphery in the 8 h WT-infected cell, and a pre-replication site (red) next to a small Hsc70 focus site (merge) in the 12 h dLeu-infected cell. In the right panels, cells were stained with anti-Hsc70 antibody and anti-RNAP II antibody H5, which recognizes phospho-serine-2 RNAP II CTD. This antibody also cross-reacts with a phospho-epitope in splicing SR proteins [11], [40]. The arrow points to an Hsc70 focus site (green) adjacent to a splicing speckle (red).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001491-g007: Hsc70 Nuclear Sequestration Correlates with the Loss of Phospho-serine 2 Form of RNAP II CTD in Fully Formed Replication Compartments.Vero cells were mock-infected or infected with WT HSV-1, dLeu or n406 for the times indicated. In the left hand panels, cells were stained with anti-RNAP II antibody, H14, which recognizes phospho-serine 5 RNAP II CTD, and anti-Hsc70 antibody. Arrows show a replication compartment with an Hsc70 focus site at the periphery in the 8 h WT-infected cell, and a pre-replication site (red) next to a small Hsc70 focus site (merge) in the 12 h dLeu-infected cell. In the right panels, cells were stained with anti-Hsc70 antibody and anti-RNAP II antibody H5, which recognizes phospho-serine-2 RNAP II CTD. This antibody also cross-reacts with a phospho-epitope in splicing SR proteins [11], [40]. The arrow points to an Hsc70 focus site (green) adjacent to a splicing speckle (red).
Mentions: To monitor relocalization of RNAP II to viral replication compartments, staining was performed with monoclonal antibody H14, which recognizes the serine-5 phosphorylated form of RNAP II CTD found in the initiation complex [38], [39]. Serine-5 phosphorylated RNAP II was recruited to structures resembling viral replication compartments by 8 h after infection in WT HSV-1-infected cells, and Hsc70 foci formed at the periphery of these structures (Figure 7, left panels). To show that these structures were indeed replication compartments, H14 stained cells were also stained with antibody to ICP4 (Figure S1). It is clear that serine-5 RNAP II stained by H14 antibody colocalized with ICP4-marked replication compartments. In contrast, in dLeu and n406 infections, H14 staining was similar to mock infected cells at 8 h for dLeu and 6 h for n406-infected cells (Figure 7, left panels). It was not until later times with these mutants that small Hsc70 foci were seen to form along with small compartments that stained with H14, and which presumably represent viral pre-replication sites (Figure 7, left panels and Figure S1).

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