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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

Dominant Negative Mutant Hsc70 K71M Prevents Proteasomal Degradation of Phospho-Serine 2 RNAP II CTD.A) RSF cells were transfected with vector alone (-), Myc-tagged Hsc70 or Myc-tagged Hsc70K71M and 24 h later were either mock-infected or were infected with WT HSV-1 or 27-LacZ. Nuclear extracts were prepared 8 h after infection. Western blots were probed with antibody H5 to detect the phospho-serine 2 form of RNAP II; anti-myc antibody to detect myc-tagged Hsc70; anti-ICP27 antibody to monitor ICP27 expression, and anti-YY1 antibody as a loading control. YY1 is a cellular transcription factor. B) HeLa cells were either mock-infected or infected with WT HSV-1. MG132 (50 µM) was added at the times indicated and nuclear extracts were prepared at 8 after infection. Western blots were probed with H5 antibody to detect RNAP II phospho-serine 2; ARNA3 antibody, which recognizes all forms of RNAP II, both phosphorylated and unphosphorylated; anti-ICP27 antibody, and anti-YY1 antibody. C) RSF cells were transfected with empty vector alone (control), myc-tagged Hsc70 or myc-tagged Hsc70K71M as indicated, and were infected 24 h later with WT HSV-1. Nuclear extracts were prepared 8 h after infection, and samples were fractionated directly by SDS-PAGE for Western blot analysis (left panel, NE) or were immunoprecipitated with anti-ubiquitin antibody (right panel, IP- anti-Ub). Immunoprecipitated complexes were fractionated by SDS-PAGE and the Western blot was probed with anti-RNAP II antibody ARNA3. Arrows point to slower migrating ubiquitinated forms of RNAP II. D) RSF cells were transfected with empty vector alone (control), myc-tagged Hsc70 or myc-tagged Hsc70K71M and were infected 24 h later with WT HSV-1. Cells were harvested 24 h after infection and viral titers were determined by plaque assays.
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pone-0001491-g010: Dominant Negative Mutant Hsc70 K71M Prevents Proteasomal Degradation of Phospho-Serine 2 RNAP II CTD.A) RSF cells were transfected with vector alone (-), Myc-tagged Hsc70 or Myc-tagged Hsc70K71M and 24 h later were either mock-infected or were infected with WT HSV-1 or 27-LacZ. Nuclear extracts were prepared 8 h after infection. Western blots were probed with antibody H5 to detect the phospho-serine 2 form of RNAP II; anti-myc antibody to detect myc-tagged Hsc70; anti-ICP27 antibody to monitor ICP27 expression, and anti-YY1 antibody as a loading control. YY1 is a cellular transcription factor. B) HeLa cells were either mock-infected or infected with WT HSV-1. MG132 (50 µM) was added at the times indicated and nuclear extracts were prepared at 8 after infection. Western blots were probed with H5 antibody to detect RNAP II phospho-serine 2; ARNA3 antibody, which recognizes all forms of RNAP II, both phosphorylated and unphosphorylated; anti-ICP27 antibody, and anti-YY1 antibody. C) RSF cells were transfected with empty vector alone (control), myc-tagged Hsc70 or myc-tagged Hsc70K71M as indicated, and were infected 24 h later with WT HSV-1. Nuclear extracts were prepared 8 h after infection, and samples were fractionated directly by SDS-PAGE for Western blot analysis (left panel, NE) or were immunoprecipitated with anti-ubiquitin antibody (right panel, IP- anti-Ub). Immunoprecipitated complexes were fractionated by SDS-PAGE and the Western blot was probed with anti-RNAP II antibody ARNA3. Arrows point to slower migrating ubiquitinated forms of RNAP II. D) RSF cells were transfected with empty vector alone (control), myc-tagged Hsc70 or myc-tagged Hsc70K71M and were infected 24 h later with WT HSV-1. Cells were harvested 24 h after infection and viral titers were determined by plaque assays.

Mentions: To further explore the apparent inhibition of RNAP II degradation by the Hsc70 dominant negative mutant, Western blot analysis was performed on nuclear extracts from cells that were transfected with GFP or GFP-Hsc70 as controls, or with GFP-Hsc70 K71M. Cells were subsequently mock-infected or were infected with WT HSV-1 or 27-LacZ. Blots were probed with antibody H5 to detect the serine-2 form of RNAP II CTD. Note that in Western blots, cross-reactivity of H5 with SR proteins does not prevent detection of serine-2 RNAP II CTD because its apparent molecular weight is greater than 180 kDa, whereas, the cross-reacting SR proteins are around 35 kDa. Levels of serine-2 phosphorylated RNAP II CTD were similar in all samples from mock infected cells and in all samples from cells infected with 27-LacZ (Figure 10A). This is in accord with our previous finding that there is no discernible loss of the serine-2 form in mock infected cells, and that degradation of RNAP II occurs to a lesser extent and at much later times in 27-LacZ infected cells, which are severely defective in viral transcription [11], [43]. On the other hand, there was a marked reduction in the serine-2 form of RNAP II in WT HSV-1 infected cells, such that it was not detectable in cells transfected with GFP alone and was barely detectable in cells transfected with GFP-Hsc70, indicating that proteasomal degradation had occurred. The dominant negative Hsc70 K71M interfered with RNAP II degradation, because levels of the serine-2 phospho-form that were detected were significantly higher compared with the controls (Figure 10A). The rescue of RNAP II from degradation by Hsc70 K71M was on par with the addition of MG132 (Figure 10B). When MG132 was added to WT HSV-1 infected cells at different times after infection, there was inhibition of phosphoserine-2 RNAP II and total RNAP II degradation, with the greatest effects seen with the early addition of the drug before robust viral transcription and replication (Figure 10B).


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)

Dominant Negative Mutant Hsc70 K71M Prevents Proteasomal Degradation of Phospho-Serine 2 RNAP II CTD.A) RSF cells were transfected with vector alone (-), Myc-tagged Hsc70 or Myc-tagged Hsc70K71M and 24 h later were either mock-infected or were infected with WT HSV-1 or 27-LacZ. Nuclear extracts were prepared 8 h after infection. Western blots were probed with antibody H5 to detect the phospho-serine 2 form of RNAP II; anti-myc antibody to detect myc-tagged Hsc70; anti-ICP27 antibody to monitor ICP27 expression, and anti-YY1 antibody as a loading control. YY1 is a cellular transcription factor. B) HeLa cells were either mock-infected or infected with WT HSV-1. MG132 (50 µM) was added at the times indicated and nuclear extracts were prepared at 8 after infection. Western blots were probed with H5 antibody to detect RNAP II phospho-serine 2; ARNA3 antibody, which recognizes all forms of RNAP II, both phosphorylated and unphosphorylated; anti-ICP27 antibody, and anti-YY1 antibody. C) RSF cells were transfected with empty vector alone (control), myc-tagged Hsc70 or myc-tagged Hsc70K71M as indicated, and were infected 24 h later with WT HSV-1. Nuclear extracts were prepared 8 h after infection, and samples were fractionated directly by SDS-PAGE for Western blot analysis (left panel, NE) or were immunoprecipitated with anti-ubiquitin antibody (right panel, IP- anti-Ub). Immunoprecipitated complexes were fractionated by SDS-PAGE and the Western blot was probed with anti-RNAP II antibody ARNA3. Arrows point to slower migrating ubiquitinated forms of RNAP II. D) RSF cells were transfected with empty vector alone (control), myc-tagged Hsc70 or myc-tagged Hsc70K71M and were infected 24 h later with WT HSV-1. Cells were harvested 24 h after infection and viral titers were determined by plaque assays.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001491-g010: Dominant Negative Mutant Hsc70 K71M Prevents Proteasomal Degradation of Phospho-Serine 2 RNAP II CTD.A) RSF cells were transfected with vector alone (-), Myc-tagged Hsc70 or Myc-tagged Hsc70K71M and 24 h later were either mock-infected or were infected with WT HSV-1 or 27-LacZ. Nuclear extracts were prepared 8 h after infection. Western blots were probed with antibody H5 to detect the phospho-serine 2 form of RNAP II; anti-myc antibody to detect myc-tagged Hsc70; anti-ICP27 antibody to monitor ICP27 expression, and anti-YY1 antibody as a loading control. YY1 is a cellular transcription factor. B) HeLa cells were either mock-infected or infected with WT HSV-1. MG132 (50 µM) was added at the times indicated and nuclear extracts were prepared at 8 after infection. Western blots were probed with H5 antibody to detect RNAP II phospho-serine 2; ARNA3 antibody, which recognizes all forms of RNAP II, both phosphorylated and unphosphorylated; anti-ICP27 antibody, and anti-YY1 antibody. C) RSF cells were transfected with empty vector alone (control), myc-tagged Hsc70 or myc-tagged Hsc70K71M as indicated, and were infected 24 h later with WT HSV-1. Nuclear extracts were prepared 8 h after infection, and samples were fractionated directly by SDS-PAGE for Western blot analysis (left panel, NE) or were immunoprecipitated with anti-ubiquitin antibody (right panel, IP- anti-Ub). Immunoprecipitated complexes were fractionated by SDS-PAGE and the Western blot was probed with anti-RNAP II antibody ARNA3. Arrows point to slower migrating ubiquitinated forms of RNAP II. D) RSF cells were transfected with empty vector alone (control), myc-tagged Hsc70 or myc-tagged Hsc70K71M and were infected 24 h later with WT HSV-1. Cells were harvested 24 h after infection and viral titers were determined by plaque assays.
Mentions: To further explore the apparent inhibition of RNAP II degradation by the Hsc70 dominant negative mutant, Western blot analysis was performed on nuclear extracts from cells that were transfected with GFP or GFP-Hsc70 as controls, or with GFP-Hsc70 K71M. Cells were subsequently mock-infected or were infected with WT HSV-1 or 27-LacZ. Blots were probed with antibody H5 to detect the serine-2 form of RNAP II CTD. Note that in Western blots, cross-reactivity of H5 with SR proteins does not prevent detection of serine-2 RNAP II CTD because its apparent molecular weight is greater than 180 kDa, whereas, the cross-reacting SR proteins are around 35 kDa. Levels of serine-2 phosphorylated RNAP II CTD were similar in all samples from mock infected cells and in all samples from cells infected with 27-LacZ (Figure 10A). This is in accord with our previous finding that there is no discernible loss of the serine-2 form in mock infected cells, and that degradation of RNAP II occurs to a lesser extent and at much later times in 27-LacZ infected cells, which are severely defective in viral transcription [11], [43]. On the other hand, there was a marked reduction in the serine-2 form of RNAP II in WT HSV-1 infected cells, such that it was not detectable in cells transfected with GFP alone and was barely detectable in cells transfected with GFP-Hsc70, indicating that proteasomal degradation had occurred. The dominant negative Hsc70 K71M interfered with RNAP II degradation, because levels of the serine-2 phospho-form that were detected were significantly higher compared with the controls (Figure 10A). The rescue of RNAP II from degradation by Hsc70 K71M was on par with the addition of MG132 (Figure 10B). When MG132 was added to WT HSV-1 infected cells at different times after infection, there was inhibition of phosphoserine-2 RNAP II and total RNAP II degradation, with the greatest effects seen with the early addition of the drug before robust viral transcription and replication (Figure 10B).

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