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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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The N- and C-termini of ICP27 Are Required for Interaction with Hsc70 during Infection.A) HeLa cells were either mock-infected or infected with WT HSV-1 or ICP27 mutants as indicated. At 6 h after infection, nuclear extracts were prepared. Expression of endogenous Hsc70 in mock-infected cells and HSV-1 WT- and mutant-infected cells is shown by Western blot analysis of samples from nuclear extracts in the top panel. Immunoprecipitations with anti-ICP27 antibody followed by Western blot analysis with anti-Hsc70 antibody are shown in the middle panel. The bottom panel shows a Western blot of anti-ICP27 immunoprecipitated samples probed with anti-ICP27 antibody to show ICP27 expression. Asterisks (*) mark WT and mutant ICP27 protein bands. The + signs mark heavy chain IgG from the immunoprecipitations. B) Reverse immunoprecipitations were performed on samples from the nuclear extracts described above. The top panel shows the expression of WT and mutant ICP27 in the nuclear extracts. Immunoprecipitation of each sample was performed with anti-Hsc70 antibody and Western blot analysis was performed by probing with anti-ICP27 antibody, as shown in the middle panel. The + sign marks heavy chain IgG from the immunoprecipitation. The bottom panel shows a Western blot of anti-Hsc70 immunoprecipitated samples probed with anti-Hsc70 antibody. C) A schematic diagram of the ICP27 coding region is shown in the left panel with the positions of the deletions illustrated by dotted lines. The table in the right panel describes the residues deleted or mutated in each mutant virus.
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pone-0001491-g003: The N- and C-termini of ICP27 Are Required for Interaction with Hsc70 during Infection.A) HeLa cells were either mock-infected or infected with WT HSV-1 or ICP27 mutants as indicated. At 6 h after infection, nuclear extracts were prepared. Expression of endogenous Hsc70 in mock-infected cells and HSV-1 WT- and mutant-infected cells is shown by Western blot analysis of samples from nuclear extracts in the top panel. Immunoprecipitations with anti-ICP27 antibody followed by Western blot analysis with anti-Hsc70 antibody are shown in the middle panel. The bottom panel shows a Western blot of anti-ICP27 immunoprecipitated samples probed with anti-ICP27 antibody to show ICP27 expression. Asterisks (*) mark WT and mutant ICP27 protein bands. The + signs mark heavy chain IgG from the immunoprecipitations. B) Reverse immunoprecipitations were performed on samples from the nuclear extracts described above. The top panel shows the expression of WT and mutant ICP27 in the nuclear extracts. Immunoprecipitation of each sample was performed with anti-Hsc70 antibody and Western blot analysis was performed by probing with anti-ICP27 antibody, as shown in the middle panel. The + sign marks heavy chain IgG from the immunoprecipitation. The bottom panel shows a Western blot of anti-Hsc70 immunoprecipitated samples probed with anti-Hsc70 antibody. C) A schematic diagram of the ICP27 coding region is shown in the left panel with the positions of the deletions illustrated by dotted lines. The table in the right panel describes the residues deleted or mutated in each mutant virus.

Mentions: To confirm the interaction of ICP27 with Hsc70 in virus-infected cells, co-immunoprecipitation experiments were performed. Cells were infected with WT HSV-1 strain KOS or with ICP27 mutant viruses as indicated (Figure 3) and at 6 hours after infection, cell lysates were immunoprecipitated with anti-ICP27 monoclonal antibody. Western blot analysis was performed with anti-Hsc70 antibody. Hsc70 was co-immunoprecipitated with ICP27 in WT HSV-1-infected cells and in cells infected with mutants d1-2, d5-6 and n504 (Figure 3A). Hsc70 was not detected in immunoprecipitated samples from cells infected with the mutant 27-LacZ, the N-terminal mutant dLeu, nor the C-terminal mutant n406. Similarly, in the reverse immunoprecipitation experiment performed with anti-Hsc70 antibody, ICP27 was not detected in Hsc70 immunoprecipitated samples from 27-LacZ-, dLeu- and n406-infected cells (Figure 3B). We conclude that ICP27 interacts with Hsc70 in virus-infected cells and N-terminal residues 6-19 and C-terminal residues 406 to 504 must be intact for this interaction to occur.


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)

The N- and C-termini of ICP27 Are Required for Interaction with Hsc70 during Infection.A) HeLa cells were either mock-infected or infected with WT HSV-1 or ICP27 mutants as indicated. At 6 h after infection, nuclear extracts were prepared. Expression of endogenous Hsc70 in mock-infected cells and HSV-1 WT- and mutant-infected cells is shown by Western blot analysis of samples from nuclear extracts in the top panel. Immunoprecipitations with anti-ICP27 antibody followed by Western blot analysis with anti-Hsc70 antibody are shown in the middle panel. The bottom panel shows a Western blot of anti-ICP27 immunoprecipitated samples probed with anti-ICP27 antibody to show ICP27 expression. Asterisks (*) mark WT and mutant ICP27 protein bands. The + signs mark heavy chain IgG from the immunoprecipitations. B) Reverse immunoprecipitations were performed on samples from the nuclear extracts described above. The top panel shows the expression of WT and mutant ICP27 in the nuclear extracts. Immunoprecipitation of each sample was performed with anti-Hsc70 antibody and Western blot analysis was performed by probing with anti-ICP27 antibody, as shown in the middle panel. The + sign marks heavy chain IgG from the immunoprecipitation. The bottom panel shows a Western blot of anti-Hsc70 immunoprecipitated samples probed with anti-Hsc70 antibody. C) A schematic diagram of the ICP27 coding region is shown in the left panel with the positions of the deletions illustrated by dotted lines. The table in the right panel describes the residues deleted or mutated in each mutant virus.
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Related In: Results  -  Collection

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pone-0001491-g003: The N- and C-termini of ICP27 Are Required for Interaction with Hsc70 during Infection.A) HeLa cells were either mock-infected or infected with WT HSV-1 or ICP27 mutants as indicated. At 6 h after infection, nuclear extracts were prepared. Expression of endogenous Hsc70 in mock-infected cells and HSV-1 WT- and mutant-infected cells is shown by Western blot analysis of samples from nuclear extracts in the top panel. Immunoprecipitations with anti-ICP27 antibody followed by Western blot analysis with anti-Hsc70 antibody are shown in the middle panel. The bottom panel shows a Western blot of anti-ICP27 immunoprecipitated samples probed with anti-ICP27 antibody to show ICP27 expression. Asterisks (*) mark WT and mutant ICP27 protein bands. The + signs mark heavy chain IgG from the immunoprecipitations. B) Reverse immunoprecipitations were performed on samples from the nuclear extracts described above. The top panel shows the expression of WT and mutant ICP27 in the nuclear extracts. Immunoprecipitation of each sample was performed with anti-Hsc70 antibody and Western blot analysis was performed by probing with anti-ICP27 antibody, as shown in the middle panel. The + sign marks heavy chain IgG from the immunoprecipitation. The bottom panel shows a Western blot of anti-Hsc70 immunoprecipitated samples probed with anti-Hsc70 antibody. C) A schematic diagram of the ICP27 coding region is shown in the left panel with the positions of the deletions illustrated by dotted lines. The table in the right panel describes the residues deleted or mutated in each mutant virus.
Mentions: To confirm the interaction of ICP27 with Hsc70 in virus-infected cells, co-immunoprecipitation experiments were performed. Cells were infected with WT HSV-1 strain KOS or with ICP27 mutant viruses as indicated (Figure 3) and at 6 hours after infection, cell lysates were immunoprecipitated with anti-ICP27 monoclonal antibody. Western blot analysis was performed with anti-Hsc70 antibody. Hsc70 was co-immunoprecipitated with ICP27 in WT HSV-1-infected cells and in cells infected with mutants d1-2, d5-6 and n504 (Figure 3A). Hsc70 was not detected in immunoprecipitated samples from cells infected with the mutant 27-LacZ, the N-terminal mutant dLeu, nor the C-terminal mutant n406. Similarly, in the reverse immunoprecipitation experiment performed with anti-Hsc70 antibody, ICP27 was not detected in Hsc70 immunoprecipitated samples from 27-LacZ-, dLeu- and n406-infected cells (Figure 3B). We conclude that ICP27 interacts with Hsc70 in virus-infected cells and N-terminal residues 6-19 and C-terminal residues 406 to 504 must be intact for this interaction to occur.

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