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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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Detergent resistant interaction of the GMPs with cellular chromatin.Representative images illustrating the nuclear fluorescence of U2OS cells transiently expressing the indicted GFP-tagged GMPs without or with treatment for 5 min in the presence of 0.5% Triton X-100 before fixation. (a) The nuclei are visualized by staining with DAPI. Digital images were captured using a LEITZ-DMRB fluorescence microscope equipped with a CCD camera. (b) The fluorescence intensity was quantified in 100 nuclei from each condition using the ImageJ software. The percentage of residual fluorescence was calculated as (mean fluorescence treated cells/mean fluorescence untreated cells) ×100. The mean ± SE of three experiments is shown in the figure. *** = p<0.001.
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pone-0062783-g002: Detergent resistant interaction of the GMPs with cellular chromatin.Representative images illustrating the nuclear fluorescence of U2OS cells transiently expressing the indicted GFP-tagged GMPs without or with treatment for 5 min in the presence of 0.5% Triton X-100 before fixation. (a) The nuclei are visualized by staining with DAPI. Digital images were captured using a LEITZ-DMRB fluorescence microscope equipped with a CCD camera. (b) The fluorescence intensity was quantified in 100 nuclei from each condition using the ImageJ software. The percentage of residual fluorescence was calculated as (mean fluorescence treated cells/mean fluorescence untreated cells) ×100. The mean ± SE of three experiments is shown in the figure. *** = p<0.001.

Mentions: In view of the different chromatin targeting modules of LCV and RHV encoded GMPs, we then asked whether this might influence the strength of interaction with cellular chromatin. We have previously shown that the interaction of EBNA1 with cellular chromatin is resistant to extraction with 0.5% Triton X-100, which prevents proteasomal degradation and contributes to the very slow turnover of the protein [8]. In order to test whether this property is shared by other GMPs, aliquots of U2OS cells were seeded on cover slides 24 hrs after transfection and the fluorescence intensity of cells grown for one more day was quantified with or without prior extraction with 0.5% Triton X-100 for 5 minutes (Figure 2a). As expected, the treatment resulted in loss of fluorescence in cells expressing GFP-NLS that does not interact with chromatin, whereas cells expressing the GFP-GMPs retained between 75% and up to 100% of the fluorescence detected in untreated cells (Figure 2b). Thus, all GMPs establish strong interactions with cellular chromatin.


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

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

Detergent resistant interaction of the GMPs with cellular chromatin.Representative images illustrating the nuclear fluorescence of U2OS cells transiently expressing the indicted GFP-tagged GMPs without or with treatment for 5 min in the presence of 0.5% Triton X-100 before fixation. (a) The nuclei are visualized by staining with DAPI. Digital images were captured using a LEITZ-DMRB fluorescence microscope equipped with a CCD camera. (b) The fluorescence intensity was quantified in 100 nuclei from each condition using the ImageJ software. The percentage of residual fluorescence was calculated as (mean fluorescence treated cells/mean fluorescence untreated cells) ×100. The mean ± SE of three experiments is shown in the figure. *** = p<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646995&req=5

pone-0062783-g002: Detergent resistant interaction of the GMPs with cellular chromatin.Representative images illustrating the nuclear fluorescence of U2OS cells transiently expressing the indicted GFP-tagged GMPs without or with treatment for 5 min in the presence of 0.5% Triton X-100 before fixation. (a) The nuclei are visualized by staining with DAPI. Digital images were captured using a LEITZ-DMRB fluorescence microscope equipped with a CCD camera. (b) The fluorescence intensity was quantified in 100 nuclei from each condition using the ImageJ software. The percentage of residual fluorescence was calculated as (mean fluorescence treated cells/mean fluorescence untreated cells) ×100. The mean ± SE of three experiments is shown in the figure. *** = p<0.001.
Mentions: In view of the different chromatin targeting modules of LCV and RHV encoded GMPs, we then asked whether this might influence the strength of interaction with cellular chromatin. We have previously shown that the interaction of EBNA1 with cellular chromatin is resistant to extraction with 0.5% Triton X-100, which prevents proteasomal degradation and contributes to the very slow turnover of the protein [8]. In order to test whether this property is shared by other GMPs, aliquots of U2OS cells were seeded on cover slides 24 hrs after transfection and the fluorescence intensity of cells grown for one more day was quantified with or without prior extraction with 0.5% Triton X-100 for 5 minutes (Figure 2a). As expected, the treatment resulted in loss of fluorescence in cells expressing GFP-NLS that does not interact with chromatin, whereas cells expressing the GFP-GMPs retained between 75% and up to 100% of the fluorescence detected in untreated cells (Figure 2b). Thus, all GMPs establish strong interactions with cellular chromatin.

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