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A Method for Visualization of Incoming Adenovirus Chromatin Complexes in Fixed and Living Cells.

Komatsu T, Dacheux D, Kreppel F, Nagata K, Wodrich H - PLoS ONE (2015)

Bottom Line: Furthermore, we describe the development of a novel imaging system that uses Template Activating Factor-I (TAF-I/SET), a cellular chromatin protein tightly bound to protein VII upon infection.This setup allows us not only to rapidly visualize protein VII foci in fixed cells but also to monitor their movement in living cells.These powerful tools can provide novel insights into the spatio-temporal regulation of incoming adenoviral chromatin complexes.

View Article: PubMed Central - PubMed

Affiliation: Microbiologie Fondamentale et Pathogénicité, MFP CNRS UMR 5234, Université de Bordeaux, Bordeaux 33076, France; Department of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Japan.

ABSTRACT
Inside the adenovirus virion, the genome forms a chromatin-like structure with viral basic core proteins. Core protein VII is the major DNA binding protein and was shown to remain associated with viral genomes upon virus entry even after nuclear delivery. It has been suggested that protein VII plays a regulatory role in viral gene expression and is a functional component of viral chromatin complexes in host cells. As such, protein VII could be used as a maker to track adenoviral chromatin complexes in vivo. In this study, we characterize a new monoclonal antibody against protein VII that stains incoming viral chromatin complexes following nuclear import. Furthermore, we describe the development of a novel imaging system that uses Template Activating Factor-I (TAF-I/SET), a cellular chromatin protein tightly bound to protein VII upon infection. This setup allows us not only to rapidly visualize protein VII foci in fixed cells but also to monitor their movement in living cells. These powerful tools can provide novel insights into the spatio-temporal regulation of incoming adenoviral chromatin complexes.

No MeSH data available.


Related in: MedlinePlus

IF analyses using EGFP-TAF-Iβ.(A) IF analyses with anti-TAF-Iβ antibody. U2OS cells were either mock-infected or infected with Ad5. At 3 hpi, cells were subjected to IF analyses using mouse anti-TAF-Iβ (green) and rat anti-protein VII antibodies (red). (B) IF analyses with EGFP-TAF-Iβ. U2OS cells were transiently transfected with the expression vector for EGFP-TAF-Iβ (green) and at 24 hpt (hours post transfection) were either mock-infected or infected with Ad5. At 3 hpi, cells were either immediately fixed (first and second rows) or pre-extracted with Triton X-100 and then fixed (third row, + Triton) and subjected to IF analyses using rat anti-protein VII antibody (red). Nuclear shapes are indicated by dashed lines. (C) IF analyses with TAF-IβPME mutant. U2OS cells were transiently transfected with the expression vectors for either EGFP-TAF-IβWT (left) or PME mutant (right panels), together with the one for histone H2B-tdiRFP (gray), and at 24 hpt IF analyses were carried out as described above.
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pone.0137102.g003: IF analyses using EGFP-TAF-Iβ.(A) IF analyses with anti-TAF-Iβ antibody. U2OS cells were either mock-infected or infected with Ad5. At 3 hpi, cells were subjected to IF analyses using mouse anti-TAF-Iβ (green) and rat anti-protein VII antibodies (red). (B) IF analyses with EGFP-TAF-Iβ. U2OS cells were transiently transfected with the expression vector for EGFP-TAF-Iβ (green) and at 24 hpt (hours post transfection) were either mock-infected or infected with Ad5. At 3 hpi, cells were either immediately fixed (first and second rows) or pre-extracted with Triton X-100 and then fixed (third row, + Triton) and subjected to IF analyses using rat anti-protein VII antibody (red). Nuclear shapes are indicated by dashed lines. (C) IF analyses with TAF-IβPME mutant. U2OS cells were transiently transfected with the expression vectors for either EGFP-TAF-IβWT (left) or PME mutant (right panels), together with the one for histone H2B-tdiRFP (gray), and at 24 hpt IF analyses were carried out as described above.

Mentions: The cellular protein TAF-I (Fig 3 and S2 Dataset), which co-localizes with protein VII in the nucleus upon Ad infection in HeLa cells [16], was then characterized. The endogenous TAF-Iβ co-localized with protein VII puncta in U2OS cells (Fig 3A). We reasoned that if fluorescent protein-tagged TAF-Iβ behaves like endogenous one, it could be an alternative marker for protein VII puncta in both fixed and living cells. We carried out IF analyses using EGFP-tagged TAF-Iβ and anti-protein VII antibodies (Fig 3B). Similar to the endogenous protein, EGFP-TAF-Iβ showed an even nuclear localization in uninfected cells (Fig 3B, first row). However, we could not observe clear overlap with protein VII puncta in infected cells (Fig 3B, second row) unless cells were pre-extracted using detergent prior to fixation (Fig 3B, third row), suggesting that EGFP-TAF-I formed detergent-resistant complexes with genome-bound protein VII. To verify the complex formation with protein VII, we performed the same IF analyses using a TAF-Iβ dimerization mutant TAF-IβPME [25]. Dimerization was previously shown to be critical for cellular and viral chromatin remodeling activity [25,26]. Cells were first transfected with the expression vectors for either EGFP-tagged wildtype TAF-Iβ (WT) or the PME mutant, together with an expression vector for tdiRFP-tagged histone H2B as a transfection marker that was resistant to pre-extraction, and then IF analyses were carried out (Fig 3C). Pre-extraction with infected cells resulted in nuclear puncta of TAF-IβWT but not PME (Fig 3C, third row), confirming a functional interaction with protein VII. As EGFP-TAF-Iβ co-localized with protein VII almost completely (Fig 3B and 3C), our data suggests that the use of this protein allows the rapid detection of Ad chromatin complexes without further antibody staining.


A Method for Visualization of Incoming Adenovirus Chromatin Complexes in Fixed and Living Cells.

Komatsu T, Dacheux D, Kreppel F, Nagata K, Wodrich H - PLoS ONE (2015)

IF analyses using EGFP-TAF-Iβ.(A) IF analyses with anti-TAF-Iβ antibody. U2OS cells were either mock-infected or infected with Ad5. At 3 hpi, cells were subjected to IF analyses using mouse anti-TAF-Iβ (green) and rat anti-protein VII antibodies (red). (B) IF analyses with EGFP-TAF-Iβ. U2OS cells were transiently transfected with the expression vector for EGFP-TAF-Iβ (green) and at 24 hpt (hours post transfection) were either mock-infected or infected with Ad5. At 3 hpi, cells were either immediately fixed (first and second rows) or pre-extracted with Triton X-100 and then fixed (third row, + Triton) and subjected to IF analyses using rat anti-protein VII antibody (red). Nuclear shapes are indicated by dashed lines. (C) IF analyses with TAF-IβPME mutant. U2OS cells were transiently transfected with the expression vectors for either EGFP-TAF-IβWT (left) or PME mutant (right panels), together with the one for histone H2B-tdiRFP (gray), and at 24 hpt IF analyses were carried out as described above.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4557953&req=5

pone.0137102.g003: IF analyses using EGFP-TAF-Iβ.(A) IF analyses with anti-TAF-Iβ antibody. U2OS cells were either mock-infected or infected with Ad5. At 3 hpi, cells were subjected to IF analyses using mouse anti-TAF-Iβ (green) and rat anti-protein VII antibodies (red). (B) IF analyses with EGFP-TAF-Iβ. U2OS cells were transiently transfected with the expression vector for EGFP-TAF-Iβ (green) and at 24 hpt (hours post transfection) were either mock-infected or infected with Ad5. At 3 hpi, cells were either immediately fixed (first and second rows) or pre-extracted with Triton X-100 and then fixed (third row, + Triton) and subjected to IF analyses using rat anti-protein VII antibody (red). Nuclear shapes are indicated by dashed lines. (C) IF analyses with TAF-IβPME mutant. U2OS cells were transiently transfected with the expression vectors for either EGFP-TAF-IβWT (left) or PME mutant (right panels), together with the one for histone H2B-tdiRFP (gray), and at 24 hpt IF analyses were carried out as described above.
Mentions: The cellular protein TAF-I (Fig 3 and S2 Dataset), which co-localizes with protein VII in the nucleus upon Ad infection in HeLa cells [16], was then characterized. The endogenous TAF-Iβ co-localized with protein VII puncta in U2OS cells (Fig 3A). We reasoned that if fluorescent protein-tagged TAF-Iβ behaves like endogenous one, it could be an alternative marker for protein VII puncta in both fixed and living cells. We carried out IF analyses using EGFP-tagged TAF-Iβ and anti-protein VII antibodies (Fig 3B). Similar to the endogenous protein, EGFP-TAF-Iβ showed an even nuclear localization in uninfected cells (Fig 3B, first row). However, we could not observe clear overlap with protein VII puncta in infected cells (Fig 3B, second row) unless cells were pre-extracted using detergent prior to fixation (Fig 3B, third row), suggesting that EGFP-TAF-I formed detergent-resistant complexes with genome-bound protein VII. To verify the complex formation with protein VII, we performed the same IF analyses using a TAF-Iβ dimerization mutant TAF-IβPME [25]. Dimerization was previously shown to be critical for cellular and viral chromatin remodeling activity [25,26]. Cells were first transfected with the expression vectors for either EGFP-tagged wildtype TAF-Iβ (WT) or the PME mutant, together with an expression vector for tdiRFP-tagged histone H2B as a transfection marker that was resistant to pre-extraction, and then IF analyses were carried out (Fig 3C). Pre-extraction with infected cells resulted in nuclear puncta of TAF-IβWT but not PME (Fig 3C, third row), confirming a functional interaction with protein VII. As EGFP-TAF-Iβ co-localized with protein VII almost completely (Fig 3B and 3C), our data suggests that the use of this protein allows the rapid detection of Ad chromatin complexes without further antibody staining.

Bottom Line: Furthermore, we describe the development of a novel imaging system that uses Template Activating Factor-I (TAF-I/SET), a cellular chromatin protein tightly bound to protein VII upon infection.This setup allows us not only to rapidly visualize protein VII foci in fixed cells but also to monitor their movement in living cells.These powerful tools can provide novel insights into the spatio-temporal regulation of incoming adenoviral chromatin complexes.

View Article: PubMed Central - PubMed

Affiliation: Microbiologie Fondamentale et Pathogénicité, MFP CNRS UMR 5234, Université de Bordeaux, Bordeaux 33076, France; Department of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Japan.

ABSTRACT
Inside the adenovirus virion, the genome forms a chromatin-like structure with viral basic core proteins. Core protein VII is the major DNA binding protein and was shown to remain associated with viral genomes upon virus entry even after nuclear delivery. It has been suggested that protein VII plays a regulatory role in viral gene expression and is a functional component of viral chromatin complexes in host cells. As such, protein VII could be used as a maker to track adenoviral chromatin complexes in vivo. In this study, we characterize a new monoclonal antibody against protein VII that stains incoming viral chromatin complexes following nuclear import. Furthermore, we describe the development of a novel imaging system that uses Template Activating Factor-I (TAF-I/SET), a cellular chromatin protein tightly bound to protein VII upon infection. This setup allows us not only to rapidly visualize protein VII foci in fixed cells but also to monitor their movement in living cells. These powerful tools can provide novel insights into the spatio-temporal regulation of incoming adenoviral chromatin complexes.

No MeSH data available.


Related in: MedlinePlus