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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

An Hsc70 Dominant Negative Mutant Can Prevent RNAP II Phospho-serine 2 Degradation and Hsc70 Focus Formation.A) Vero cells were transfected with pGFP-Hsc70 and were subsequently infected with WT HSV-1 for 8 h. Cells were stained with H5, anti-ICP4 or anti-ICP8 antibodies. GFP fluorescence was viewed directly. Arrows point to Hsc70 foci. B) Vero cells were transfected with the dominant negative mutant GFP-Hsc70K71M and then infected with WT HSV-1 for 8 h. Cells were stained with H5, anti-ICP4 or anti-ICP8 antibodies. Arrows point to GFP Hsc70K71M expressing cells showing diffuse nuclear staining with H5, and to pre-replication sites in cells stained for ICP4 and ICP8. The yellow arrows in the ICP4 and ICP8 stained panels point to a fully formed replication compartment.
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pone-0001491-g009: An Hsc70 Dominant Negative Mutant Can Prevent RNAP II Phospho-serine 2 Degradation and Hsc70 Focus Formation.A) Vero cells were transfected with pGFP-Hsc70 and were subsequently infected with WT HSV-1 for 8 h. Cells were stained with H5, anti-ICP4 or anti-ICP8 antibodies. GFP fluorescence was viewed directly. Arrows point to Hsc70 foci. B) Vero cells were transfected with the dominant negative mutant GFP-Hsc70K71M and then infected with WT HSV-1 for 8 h. Cells were stained with H5, anti-ICP4 or anti-ICP8 antibodies. Arrows point to GFP Hsc70K71M expressing cells showing diffuse nuclear staining with H5, and to pre-replication sites in cells stained for ICP4 and ICP8. The yellow arrows in the ICP4 and ICP8 stained panels point to a fully formed replication compartment.

Mentions: To determine if Hsc70 K71M would interfere with Hsc70 focus formation during HSV-1 infection, cells transfected with GFP-Hsc70 K71M or GFP-Hsc70 were subsequently infected with WT HSV-1 for 8 h. Hsc70 localization was monitored by immunofluorescent microscopy. In cells transfected with GFP-Hsc70, nuclear focus formation was seen, along with speckled staining for H5, and replication compartment formation marked by staining for ICP4 and ICP8, a viral DNA replication protein (Figure 9A). In contrast, in cells expressing GFP-Hsc70 K71M, there was a diffuse nuclear and cytoplasmic distribution with no focus formation. Even more interesting was the staining of H5 in the cell expressing GFP-Hsc70 K71M, which remained diffuse in the nucleus rather than localizing to speckles as in the infected cells not expressing GFP-Hsc70 K71M (Figure 9B). This suggests that degradation of RNAP II was not occurring in the presence of the dominant negative mutant. Further, viral replication compartment formation was also hampered by the dominant negative Hsc70 mutant, as seen in the cells stained with ICP4 and ICP8 antibodies, and which also expressed GFP-Hsc70 K71M (Figure 9B).


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)

An Hsc70 Dominant Negative Mutant Can Prevent RNAP II Phospho-serine 2 Degradation and Hsc70 Focus Formation.A) Vero cells were transfected with pGFP-Hsc70 and were subsequently infected with WT HSV-1 for 8 h. Cells were stained with H5, anti-ICP4 or anti-ICP8 antibodies. GFP fluorescence was viewed directly. Arrows point to Hsc70 foci. B) Vero cells were transfected with the dominant negative mutant GFP-Hsc70K71M and then infected with WT HSV-1 for 8 h. Cells were stained with H5, anti-ICP4 or anti-ICP8 antibodies. Arrows point to GFP Hsc70K71M expressing cells showing diffuse nuclear staining with H5, and to pre-replication sites in cells stained for ICP4 and ICP8. The yellow arrows in the ICP4 and ICP8 stained panels point to a fully formed replication compartment.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001491-g009: An Hsc70 Dominant Negative Mutant Can Prevent RNAP II Phospho-serine 2 Degradation and Hsc70 Focus Formation.A) Vero cells were transfected with pGFP-Hsc70 and were subsequently infected with WT HSV-1 for 8 h. Cells were stained with H5, anti-ICP4 or anti-ICP8 antibodies. GFP fluorescence was viewed directly. Arrows point to Hsc70 foci. B) Vero cells were transfected with the dominant negative mutant GFP-Hsc70K71M and then infected with WT HSV-1 for 8 h. Cells were stained with H5, anti-ICP4 or anti-ICP8 antibodies. Arrows point to GFP Hsc70K71M expressing cells showing diffuse nuclear staining with H5, and to pre-replication sites in cells stained for ICP4 and ICP8. The yellow arrows in the ICP4 and ICP8 stained panels point to a fully formed replication compartment.
Mentions: To determine if Hsc70 K71M would interfere with Hsc70 focus formation during HSV-1 infection, cells transfected with GFP-Hsc70 K71M or GFP-Hsc70 were subsequently infected with WT HSV-1 for 8 h. Hsc70 localization was monitored by immunofluorescent microscopy. In cells transfected with GFP-Hsc70, nuclear focus formation was seen, along with speckled staining for H5, and replication compartment formation marked by staining for ICP4 and ICP8, a viral DNA replication protein (Figure 9A). In contrast, in cells expressing GFP-Hsc70 K71M, there was a diffuse nuclear and cytoplasmic distribution with no focus formation. Even more interesting was the staining of H5 in the cell expressing GFP-Hsc70 K71M, which remained diffuse in the nucleus rather than localizing to speckles as in the infected cells not expressing GFP-Hsc70 K71M (Figure 9B). This suggests that degradation of RNAP II was not occurring in the presence of the dominant negative mutant. Further, viral replication compartment formation was also hampered by the dominant negative Hsc70 mutant, as seen in the cells stained with ICP4 and ICP8 antibodies, and which also expressed GFP-Hsc70 K71M (Figure 9B).

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