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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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LCV- and RHV-GMPs show different mobility on chromatin.The mobility of GFP-GMP fusion proteins on interphase chromatin was assayed by FRAP in transiently transfected U2OS cells. (a) The normalized % Fluorescence recovery was calculated relative to the mean fluorescence at the time of bleaching (0% recovery) and the mean fluorescence of GFP-NLS after recovery for 25.5 sec (100% recovery). The mean ±SD of the relative fluorescence in 20 cells is shown in each graph. (b) Clustal W multiple sequence alignment of the chromatin targeting modules of LVC and RHV GMPs. LCV-GMP share highly conserved Gly-Arg repeat regions that resemble the AT-hook of HMGA proteins. LANA1, mnR1-LANA and mnR2-LANA share a highly conserved N-terminal domain that was shown to mediate the interaction of LANA1 with histone H2A–H2B. The chromatin binding domains of saLANA and muLANA have not been characterized.
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pone-0062783-g004: LCV- and RHV-GMPs show different mobility on chromatin.The mobility of GFP-GMP fusion proteins on interphase chromatin was assayed by FRAP in transiently transfected U2OS cells. (a) The normalized % Fluorescence recovery was calculated relative to the mean fluorescence at the time of bleaching (0% recovery) and the mean fluorescence of GFP-NLS after recovery for 25.5 sec (100% recovery). The mean ±SD of the relative fluorescence in 20 cells is shown in each graph. (b) Clustal W multiple sequence alignment of the chromatin targeting modules of LVC and RHV GMPs. LCV-GMP share highly conserved Gly-Arg repeat regions that resemble the AT-hook of HMGA proteins. LANA1, mnR1-LANA and mnR2-LANA share a highly conserved N-terminal domain that was shown to mediate the interaction of LANA1 with histone H2A–H2B. The chromatin binding domains of saLANA and muLANA have not been characterized.

Mentions: In order to reveal features of the interaction of GMPs with cellular DNA that may be relevant for the chromatin remodeling function, the mobility of GFP-tagged proteins was measured in the nucleus of transiently transfected U2OS cells by fluorescence recovery after photobleaching (FRAP). The efficiency of FRAP recovery of the GFP chimeras were compared to that of a control nuclear GFP (GFP-NLS) that does not bind to chromatin. In accordance with previous reports [9], [11], EBNA1 was highly mobile on chromatin with a virtually full recovery of fluorescence by the end of the observation period of 25.5 sec (Figure 4a). A comparable virtually full recovery was observed with baEBNA1 that shares 56% amino acid sequence homology with EBNA1 and a highly conserved AT-hook-like chromatin targeting module (Figure 4b). In contrast, a poorer recovery was observed in cells expressing the RHV GMPs. Only 25% to 35% of the initial fluorescence was recovered in cells expressing LANA1 and mnR2-LANA within the observation period, suggesting a very slow overall mobility and possibly the presence of a large immobile fraction (Figure 4a). The two proteins belong to different RHV subfamilies but share a highly conserved domain structure (Figure 1a), and more than 45% sequence identity in the N-terminal domain that was shown to mediate the interaction of LANA1 with cellular chromatin via binding to histone H2A and H2B [13] (Figure 4b). The remaining RHV GMPs showed intermediate recoveries of 67.7%, 69.7% and 84.4% at the end of the observation period for mnR1-LANA, muLANA and, saLANA, respectively (Figure 4a). The N-terminal domain of nmR1-LANA is very similar to that of LANA1 and mnR2-LANA (Figure 4b), suggesting that they could have similar interacting partners, whereas the N-terminal domains of saLANA and muLANA share the overall prevalence of basic amino acid residues but no sequence similarity with the corresponding regions of other RHV GMPs.


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

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

LCV- and RHV-GMPs show different mobility on chromatin.The mobility of GFP-GMP fusion proteins on interphase chromatin was assayed by FRAP in transiently transfected U2OS cells. (a) The normalized % Fluorescence recovery was calculated relative to the mean fluorescence at the time of bleaching (0% recovery) and the mean fluorescence of GFP-NLS after recovery for 25.5 sec (100% recovery). The mean ±SD of the relative fluorescence in 20 cells is shown in each graph. (b) Clustal W multiple sequence alignment of the chromatin targeting modules of LVC and RHV GMPs. LCV-GMP share highly conserved Gly-Arg repeat regions that resemble the AT-hook of HMGA proteins. LANA1, mnR1-LANA and mnR2-LANA share a highly conserved N-terminal domain that was shown to mediate the interaction of LANA1 with histone H2A–H2B. The chromatin binding domains of saLANA and muLANA have not been characterized.
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Related In: Results  -  Collection

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pone-0062783-g004: LCV- and RHV-GMPs show different mobility on chromatin.The mobility of GFP-GMP fusion proteins on interphase chromatin was assayed by FRAP in transiently transfected U2OS cells. (a) The normalized % Fluorescence recovery was calculated relative to the mean fluorescence at the time of bleaching (0% recovery) and the mean fluorescence of GFP-NLS after recovery for 25.5 sec (100% recovery). The mean ±SD of the relative fluorescence in 20 cells is shown in each graph. (b) Clustal W multiple sequence alignment of the chromatin targeting modules of LVC and RHV GMPs. LCV-GMP share highly conserved Gly-Arg repeat regions that resemble the AT-hook of HMGA proteins. LANA1, mnR1-LANA and mnR2-LANA share a highly conserved N-terminal domain that was shown to mediate the interaction of LANA1 with histone H2A–H2B. The chromatin binding domains of saLANA and muLANA have not been characterized.
Mentions: In order to reveal features of the interaction of GMPs with cellular DNA that may be relevant for the chromatin remodeling function, the mobility of GFP-tagged proteins was measured in the nucleus of transiently transfected U2OS cells by fluorescence recovery after photobleaching (FRAP). The efficiency of FRAP recovery of the GFP chimeras were compared to that of a control nuclear GFP (GFP-NLS) that does not bind to chromatin. In accordance with previous reports [9], [11], EBNA1 was highly mobile on chromatin with a virtually full recovery of fluorescence by the end of the observation period of 25.5 sec (Figure 4a). A comparable virtually full recovery was observed with baEBNA1 that shares 56% amino acid sequence homology with EBNA1 and a highly conserved AT-hook-like chromatin targeting module (Figure 4b). In contrast, a poorer recovery was observed in cells expressing the RHV GMPs. Only 25% to 35% of the initial fluorescence was recovered in cells expressing LANA1 and mnR2-LANA within the observation period, suggesting a very slow overall mobility and possibly the presence of a large immobile fraction (Figure 4a). The two proteins belong to different RHV subfamilies but share a highly conserved domain structure (Figure 1a), and more than 45% sequence identity in the N-terminal domain that was shown to mediate the interaction of LANA1 with cellular chromatin via binding to histone H2A and H2B [13] (Figure 4b). The remaining RHV GMPs showed intermediate recoveries of 67.7%, 69.7% and 84.4% at the end of the observation period for mnR1-LANA, muLANA and, saLANA, respectively (Figure 4a). The N-terminal domain of nmR1-LANA is very similar to that of LANA1 and mnR2-LANA (Figure 4b), suggesting that they could have similar interacting partners, whereas the N-terminal domains of saLANA and muLANA share the overall prevalence of basic amino acid residues but no sequence similarity with the corresponding regions of other RHV GMPs.

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