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Interaction of gamma-herpesvirus genome maintenance proteins with cellular chromatin.

Mughal N, Coppotelli G, Callegari S, Gastaldello S, Masucci MG - PLoS ONE (2013)

Bottom Line: We found that, in addition to a similar diffuse nuclear localization and comparable detergent resistant interaction with chromatin in transfected cells, all GMPs shared the capacity to promote the decondensation of heterochromatin in the A03-1 reporter cell line.While the AT-hook containing GMPs of LCVs were highly mobile, a great variability was observed among GMPs encoded by RHV, ranging from virtually immobile to significantly reduced mobility compared to LCV GMPs.Only the RHV GMPs recruited the bromo- and extra terminal domain (BET) proteins BRD2 and BRD4 to the site of chromatin remodeling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
The capacity of gamma-herpesviruses to establish lifelong infections is dependent on the expression of genome maintenance proteins (GMPs) that tether the viral episomes to cellular chromatin and allow their persistence in latently infected proliferating cells. Here we have characterized the chromatin interaction of GMPs encoded by viruses belonging to the genera Lymphocryptovirus (LCV) and Rhadinovirus (RHV). We found that, in addition to a similar diffuse nuclear localization and comparable detergent resistant interaction with chromatin in transfected cells, all GMPs shared the capacity to promote the decondensation of heterochromatin in the A03-1 reporter cell line. They differed, however, in their mobility measured by fluorescence recovery after photobleaching (FRAP), and in the capacity to recruit accessory molecules required for the chromatin remodeling function. While the AT-hook containing GMPs of LCVs were highly mobile, a great variability was observed among GMPs encoded by RHV, ranging from virtually immobile to significantly reduced mobility compared to LCV GMPs. Only the RHV GMPs recruited the bromo- and extra terminal domain (BET) proteins BRD2 and BRD4 to the site of chromatin remodeling. These findings suggest that differences in the mode of interaction with cellular chromatin may underlie different strategies adopted by these viruses for reprogramming of the host cells during latency.

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Localization of the GMPs in interphase nuclei.Schematic illustration of the domain organization of the GMPs included in the study. For nomenclature and references see Table 1. (a) Protein domains were identified as regions of high homology using Clustal W multiple sequence alignments. The C-terminal episome-binding domains are indicated in yellow and the chromatin targeting modules in blue. The Gly-Ala or Gly-Ala-Ser repeats of LCV-GMPs and the acidic repeats of RHV-GMPs are indicated in green and red, respectively. The acidic tails of LCV-GMPs are shown in pink. (b) Expression of GFP tagged GMPs in transfected U2OS cells. Representative western blot of total lysates from U2OS cells harvested 48 hrs after transfection probed with an anti-GFP antibody. (c) Nuclear localization of the transfected proteins. Representative fluorescence micrographs of NIH3T3 cells transfected with the indicated GFP-GMP fusion proteins or control GFP-NLS. DNA is visualized by DAPI staining (blue). The merged image and localization profile indicate that the GMPs do not accumulate on heterochromatin.
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pone-0062783-g001: Localization of the GMPs in interphase nuclei.Schematic illustration of the domain organization of the GMPs included in the study. For nomenclature and references see Table 1. (a) Protein domains were identified as regions of high homology using Clustal W multiple sequence alignments. The C-terminal episome-binding domains are indicated in yellow and the chromatin targeting modules in blue. The Gly-Ala or Gly-Ala-Ser repeats of LCV-GMPs and the acidic repeats of RHV-GMPs are indicated in green and red, respectively. The acidic tails of LCV-GMPs are shown in pink. (b) Expression of GFP tagged GMPs in transfected U2OS cells. Representative western blot of total lysates from U2OS cells harvested 48 hrs after transfection probed with an anti-GFP antibody. (c) Nuclear localization of the transfected proteins. Representative fluorescence micrographs of NIH3T3 cells transfected with the indicated GFP-GMP fusion proteins or control GFP-NLS. DNA is visualized by DAPI staining (blue). The merged image and localization profile indicate that the GMPs do not accumulate on heterochromatin.

Mentions: In order to characterize the chromatin binding properties of GMPs encoded by different LCVs and RHVs, we collected a representative panel of GFP-tagged recombinant proteins (Table 1). The domain organization of the GMPs included in the study is illustrated in Figure 1a. All GMPs contain relatively conserved C-terminal viral episome-binding domains (Figure 1a, yellow boxes), whereas the N-terminal chromatin targeting modules (Figure 1a, blue boxes) differ among the families. The N-terminus of LCV GMPs contains multiple Arg-Gly-Arg repeats that resemble the AT-hook of HMGA proteins [3], whereas a basic N-terminal domain is likely to be involved in the interaction of RHV GMPs with nucleosomes or nucleosome binding proteins [30]. In addition, the LCV GMPs contain internal repetitive sequences of Gly-Ala or Gly-Ala-Ser of different length (Figure 1a, green boxes), and a C-terminal acidic tails (Figure 1a, pink boxes), whereas some but not all RHV GMPs contain internal acidic repeats of different length and amino acid composition (Figure 1a, red boxes).


Interaction of gamma-herpesvirus genome maintenance proteins with cellular chromatin.

Mughal N, Coppotelli G, Callegari S, Gastaldello S, Masucci MG - PLoS ONE (2013)

Localization of the GMPs in interphase nuclei.Schematic illustration of the domain organization of the GMPs included in the study. For nomenclature and references see Table 1. (a) Protein domains were identified as regions of high homology using Clustal W multiple sequence alignments. The C-terminal episome-binding domains are indicated in yellow and the chromatin targeting modules in blue. The Gly-Ala or Gly-Ala-Ser repeats of LCV-GMPs and the acidic repeats of RHV-GMPs are indicated in green and red, respectively. The acidic tails of LCV-GMPs are shown in pink. (b) Expression of GFP tagged GMPs in transfected U2OS cells. Representative western blot of total lysates from U2OS cells harvested 48 hrs after transfection probed with an anti-GFP antibody. (c) Nuclear localization of the transfected proteins. Representative fluorescence micrographs of NIH3T3 cells transfected with the indicated GFP-GMP fusion proteins or control GFP-NLS. DNA is visualized by DAPI staining (blue). The merged image and localization profile indicate that the GMPs do not accumulate on heterochromatin.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646995&req=5

pone-0062783-g001: Localization of the GMPs in interphase nuclei.Schematic illustration of the domain organization of the GMPs included in the study. For nomenclature and references see Table 1. (a) Protein domains were identified as regions of high homology using Clustal W multiple sequence alignments. The C-terminal episome-binding domains are indicated in yellow and the chromatin targeting modules in blue. The Gly-Ala or Gly-Ala-Ser repeats of LCV-GMPs and the acidic repeats of RHV-GMPs are indicated in green and red, respectively. The acidic tails of LCV-GMPs are shown in pink. (b) Expression of GFP tagged GMPs in transfected U2OS cells. Representative western blot of total lysates from U2OS cells harvested 48 hrs after transfection probed with an anti-GFP antibody. (c) Nuclear localization of the transfected proteins. Representative fluorescence micrographs of NIH3T3 cells transfected with the indicated GFP-GMP fusion proteins or control GFP-NLS. DNA is visualized by DAPI staining (blue). The merged image and localization profile indicate that the GMPs do not accumulate on heterochromatin.
Mentions: In order to characterize the chromatin binding properties of GMPs encoded by different LCVs and RHVs, we collected a representative panel of GFP-tagged recombinant proteins (Table 1). The domain organization of the GMPs included in the study is illustrated in Figure 1a. All GMPs contain relatively conserved C-terminal viral episome-binding domains (Figure 1a, yellow boxes), whereas the N-terminal chromatin targeting modules (Figure 1a, blue boxes) differ among the families. The N-terminus of LCV GMPs contains multiple Arg-Gly-Arg repeats that resemble the AT-hook of HMGA proteins [3], whereas a basic N-terminal domain is likely to be involved in the interaction of RHV GMPs with nucleosomes or nucleosome binding proteins [30]. In addition, the LCV GMPs contain internal repetitive sequences of Gly-Ala or Gly-Ala-Ser of different length (Figure 1a, green boxes), and a C-terminal acidic tails (Figure 1a, pink boxes), whereas some but not all RHV GMPs contain internal acidic repeats of different length and amino acid composition (Figure 1a, red boxes).

Bottom Line: We found that, in addition to a similar diffuse nuclear localization and comparable detergent resistant interaction with chromatin in transfected cells, all GMPs shared the capacity to promote the decondensation of heterochromatin in the A03-1 reporter cell line.While the AT-hook containing GMPs of LCVs were highly mobile, a great variability was observed among GMPs encoded by RHV, ranging from virtually immobile to significantly reduced mobility compared to LCV GMPs.Only the RHV GMPs recruited the bromo- and extra terminal domain (BET) proteins BRD2 and BRD4 to the site of chromatin remodeling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
The capacity of gamma-herpesviruses to establish lifelong infections is dependent on the expression of genome maintenance proteins (GMPs) that tether the viral episomes to cellular chromatin and allow their persistence in latently infected proliferating cells. Here we have characterized the chromatin interaction of GMPs encoded by viruses belonging to the genera Lymphocryptovirus (LCV) and Rhadinovirus (RHV). We found that, in addition to a similar diffuse nuclear localization and comparable detergent resistant interaction with chromatin in transfected cells, all GMPs shared the capacity to promote the decondensation of heterochromatin in the A03-1 reporter cell line. They differed, however, in their mobility measured by fluorescence recovery after photobleaching (FRAP), and in the capacity to recruit accessory molecules required for the chromatin remodeling function. While the AT-hook containing GMPs of LCVs were highly mobile, a great variability was observed among GMPs encoded by RHV, ranging from virtually immobile to significantly reduced mobility compared to LCV GMPs. Only the RHV GMPs recruited the bromo- and extra terminal domain (BET) proteins BRD2 and BRD4 to the site of chromatin remodeling. These findings suggest that differences in the mode of interaction with cellular chromatin may underlie different strategies adopted by these viruses for reprogramming of the host cells during latency.

Show MeSH
Related in: MedlinePlus