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Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.

Dembowski JA, DeLuca NA - PLoS Pathog. (2015)

Bottom Line: We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins.Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes.Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND) was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics during infection and provides a comprehensive view of how HSV-1 selectively utilizes cellular resources.

No MeSH data available.


Related in: MedlinePlus

Visualization of EdU labeled HSV-1 genomes.(A) Prelabeled input viral genomes were visualized in the nucleus of infected cells 2 hpi. Vero cells were infected with wild type KOS or the UL2/UL50 mutant virus carrying unlabeled (0 μM) or prelabeled viral genomes. Labeled virus stocks were generated by growing KOS or UL2/UL50 mutant in the presence of 1.25 or 2.5 μM EdU as described in the experimental procedures. Cellular DNA was visualized by Hoechst staining, viral DNA by click chemistry with EdU, and ICP4 by immunofluorescence. Merged panels show colocalization of viral DNA with ICP4. Prelabeled KOS DNA could not be detected under these conditions. (B) Cells infected with KOS or UL2/UL50 mutant were grown in the presence of 0 or 2.5 μM EdU for 4–8 hpi. Uninfected cells were grown in the presence of EdU for 4 hours. DNA imaging was as described in (A).
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ppat.1004939.g002: Visualization of EdU labeled HSV-1 genomes.(A) Prelabeled input viral genomes were visualized in the nucleus of infected cells 2 hpi. Vero cells were infected with wild type KOS or the UL2/UL50 mutant virus carrying unlabeled (0 μM) or prelabeled viral genomes. Labeled virus stocks were generated by growing KOS or UL2/UL50 mutant in the presence of 1.25 or 2.5 μM EdU as described in the experimental procedures. Cellular DNA was visualized by Hoechst staining, viral DNA by click chemistry with EdU, and ICP4 by immunofluorescence. Merged panels show colocalization of viral DNA with ICP4. Prelabeled KOS DNA could not be detected under these conditions. (B) Cells infected with KOS or UL2/UL50 mutant were grown in the presence of 0 or 2.5 μM EdU for 4–8 hpi. Uninfected cells were grown in the presence of EdU for 4 hours. DNA imaging was as described in (A).

Mentions: To compare the relative amount of EdU incorporated into wild type and mutant genomes, we carried out viral infection and DNA imaging as outlined in Fig 1A and 1B. EdU labeled input genomes and replication compartments were tagged with Alexa Fluor 488 by click chemistry and visualized by fluorescence microscopy (Fig 2). Prelabeled UL2/UL50 mutant genomes that colocalized with the viral transcription factor, ICP4, were visualized in the nucleus of infected cells (Fig 2A). The distribution of ICP4 foci two hours after infection largely resembles that observed previously after infection with HSV-1 at a MOI of 10 PFU/cell [29]. Using these same conditions, we were unable to detect KOS genomes prelabeled with 2.5 μM EdU (Fig 2A). We also visualized viral replication compartments that colocalize with ICP4 8 hpi in both wild type KOS and UL2/UL50 mutant virus infected cells (Fig 2B). While it was possible to detect EdU labeling coinciding with ICP4 staining in KOS infected cells, significantly more was observed with the UL2/UL50 mutant. Taken together, the UL2/UL50 mutant virus incorporates more EdU into its genome during DNA replication and allows for more sensitive imaging of HSV-1 viral DNA during infection.


Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.

Dembowski JA, DeLuca NA - PLoS Pathog. (2015)

Visualization of EdU labeled HSV-1 genomes.(A) Prelabeled input viral genomes were visualized in the nucleus of infected cells 2 hpi. Vero cells were infected with wild type KOS or the UL2/UL50 mutant virus carrying unlabeled (0 μM) or prelabeled viral genomes. Labeled virus stocks were generated by growing KOS or UL2/UL50 mutant in the presence of 1.25 or 2.5 μM EdU as described in the experimental procedures. Cellular DNA was visualized by Hoechst staining, viral DNA by click chemistry with EdU, and ICP4 by immunofluorescence. Merged panels show colocalization of viral DNA with ICP4. Prelabeled KOS DNA could not be detected under these conditions. (B) Cells infected with KOS or UL2/UL50 mutant were grown in the presence of 0 or 2.5 μM EdU for 4–8 hpi. Uninfected cells were grown in the presence of EdU for 4 hours. DNA imaging was as described in (A).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446364&req=5

ppat.1004939.g002: Visualization of EdU labeled HSV-1 genomes.(A) Prelabeled input viral genomes were visualized in the nucleus of infected cells 2 hpi. Vero cells were infected with wild type KOS or the UL2/UL50 mutant virus carrying unlabeled (0 μM) or prelabeled viral genomes. Labeled virus stocks were generated by growing KOS or UL2/UL50 mutant in the presence of 1.25 or 2.5 μM EdU as described in the experimental procedures. Cellular DNA was visualized by Hoechst staining, viral DNA by click chemistry with EdU, and ICP4 by immunofluorescence. Merged panels show colocalization of viral DNA with ICP4. Prelabeled KOS DNA could not be detected under these conditions. (B) Cells infected with KOS or UL2/UL50 mutant were grown in the presence of 0 or 2.5 μM EdU for 4–8 hpi. Uninfected cells were grown in the presence of EdU for 4 hours. DNA imaging was as described in (A).
Mentions: To compare the relative amount of EdU incorporated into wild type and mutant genomes, we carried out viral infection and DNA imaging as outlined in Fig 1A and 1B. EdU labeled input genomes and replication compartments were tagged with Alexa Fluor 488 by click chemistry and visualized by fluorescence microscopy (Fig 2). Prelabeled UL2/UL50 mutant genomes that colocalized with the viral transcription factor, ICP4, were visualized in the nucleus of infected cells (Fig 2A). The distribution of ICP4 foci two hours after infection largely resembles that observed previously after infection with HSV-1 at a MOI of 10 PFU/cell [29]. Using these same conditions, we were unable to detect KOS genomes prelabeled with 2.5 μM EdU (Fig 2A). We also visualized viral replication compartments that colocalize with ICP4 8 hpi in both wild type KOS and UL2/UL50 mutant virus infected cells (Fig 2B). While it was possible to detect EdU labeling coinciding with ICP4 staining in KOS infected cells, significantly more was observed with the UL2/UL50 mutant. Taken together, the UL2/UL50 mutant virus incorporates more EdU into its genome during DNA replication and allows for more sensitive imaging of HSV-1 viral DNA during infection.

Bottom Line: We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins.Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes.Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND) was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics during infection and provides a comprehensive view of how HSV-1 selectively utilizes cellular resources.

No MeSH data available.


Related in: MedlinePlus