Recruitment of components of chromatin remodeling complexes by herpesvirus GMPs^a.

<p>a. More than 20 cells were examined in each test combination in three independent experiments. Score: <b>−</b> = recruitment to the array never observed; + =  clear recruitment to the array in all images; ± = recruitment to the array observed in some but not all images.</p

LCV- and RHV-GMPs show different mobility on chromatin.

<p>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.</p

Gamma-herpesvirus GMPs included in this study.

<p>a. LCV = Genus Lymphocryptovirus.</p><p>b. RHV = Genus Rhadinovirus.</p

Interaction of Gamma-Herpesvirus Genome Maintenance Proteins with Cellular Chromatin

<div><p>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.</p></div

Localization of the GMPs in interphase nuclei.

<p>Schematic illustration of the domain organization of the GMPs included in the study. For nomenclature and references see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062783#pone-0062783-t001" target="_blank">Table 1</a>. (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.</p

RHV-GMPs recruit BRD2 and BRD4 to the site of chromatin remodeling.

<p>Representative confocal images illustrating the nuclear fluorescence of A03-1 cells co-transfected with plasmids expressing mCherry-LacR-VP16 or mCherry-LacR-GMP fusion proteins and YFP-tagged BRD2 or BRD4. The recruitment of BRD2 and BRD4 to the site of chromatin decondensation, visualized by the accumulation of green fluorescence overlapping with the red fluorescent array, is indicated by arrows in cells expressing VP16 or the RHV-GMPs.</p

All GMPs promote chromatin decompaction.

<p>Representative confocal images illustrating the size of the LacO chromatin array in cells expressing LacR fused to the indicated GMPs. (a) A03-1 cells were transfected with plasmids encoding mCherry-LacR alone or the indicated fusion proteins and the size of the LacO array was measured 48 hrs after transfection. The LUT for the color-coding is shown on the left. Scale bar  = 2 µm. The size of the array relative to the size of the nucleus was calculated according to the formula: (area of the array/area of the nucleus x100) in more than 70 cells for each condition. The arrays size distribution is shown in the histograms on the right of the images. (b) Box plot illustrating the median and interquantile distribution of the size of the fluorescent arrays in cells expressing the different GMPs. The effect of the GMPs was comparable to that induced by the prototype viral transcativator VP16. *** = p<0.001.</p

Detergent resistant interaction of the GMPs with cellular chromatin.

<p>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.</p

Localization of GFP-tagged <i>L. monocytogenes</i> in acidic compartments.

<p><b>A</b>. Cells were infected with the GFP-tagged <i>L. monocytogenes</i> at MOI 50∶1 for 4 hours, and treated with the acidotropic dye Lysotracker Red. Cytospin centrifugation was performed and cells were fixed with 4% paraformaldehyde. Lysotracker Red positive vesicles containing GFP-labeled bacteria are indicated with arrows. Bar represents 5 µm. <b>B</b>. The percentage of Lysotracker Red positive vesicles containing GFP-labeled bacteria was quantified in two independent experiments. A minimum of 200 cells were analyzed. <b>C</b>. Scatter plot analysis showing the correlation between the GFP and Lysotracker Red fluorescence intensities in control cells and TAMs quantified by the Volocity software. A correlation coefficient value of r² equal to 1 indicates 100% bacterial localization with the lysosomal compartments.</p

TAMs express high levels of CD14.

<p>Non-stimulated monocytes and TAMs generated with Trx80 at 100 nM were analyzed for CD14 expression after 24 and 48 hours in culture. MFI: mean fluorescence intensity (mean ± SEM of six independent experiments).</p