SMARCB1 and UL114 interact <i>in vitro</i>.

<p>(<b>A</b>) <i>In vitro</i> binding analysis of HA-tagged Δ379-SMARCB1 and c-myc-tagged UL114 in ³⁵S-labeled proteins using the TNT coupled transcription/translation system. The proteins were transcribed and translated <i>in vitro</i> with ³⁵S-methionine in the translation mixture to generate radioactive labeled products from vectors pACT2-Δ379-SMARCB1 (HA-epitope) and pGBKT7-UL114 (c-myc epitope). The translated Δ379-SMARCB1-HA and UL114-c-myc were immunoprecipitated with either anti-HA or anti-c-myc-antibodies and eluted from the Protein G beads. Samples were subjected to 8% SDS-PAGE and PhosphoImaging. Lane 1: UL114-c-myc+c-myc antibody. Lane 2: Δ379-SMARCB1-HA+HA-antibody. Lane 3: Δ379-SMARCB1-HA+UL114-c-myc+HA-antibody. Lane 4: Δ379-SMARCB1-HA+UL114-c-myc+c-myc antibody. Lane 5: UL114-c-myc+HA-antibody. Lane 6: Δ379-SMARCB1-HA+c-myc-antibody. (^.......) indicates that samples were run on the same gel and (——) indicates that samples were run on a different gel. (<b>B</b>) and (<b>C</b>) GST pull-down assays to detect the interaction of SMARCB1 and UL114. (<b>B</b>) Purified GST-SMARCB1 or crude extract of <i>E. coli</i> cells over-expressing GST were incubated with purified UL114. The GST pull-down products were immunoblotted with anti-SMARCB1, anti-UL114 and anti-GST. Lane 1: GST-extract+UL114. Lane 2: GST-SMARCB1+UL114. Lane 3: purified GST-SMARCB1 (2 µg, 10% of input). Lane 4: Purified UL114 (1 µg, 5% of input). Note that spontaneous cleavage of GST occurred in the GST-SMARCB1 protein sample (Lanes 2 and 3). (<b>C</b>) Purified GST-SMARCB1 or crude extract of <i>E. coli</i> cells over-expressing GST were incubated with lysates of HCMV-infected cells. The GST pull-down products were immunoblotted with anti-SMARCB1, anti-UL114, anti-GST and anti-UL57. Lane 1: GST-extract+HCMV lysate. Lane 2: GST-SMARCB1+HCMV lysate. Lane 3: purified GST-SMARCB1 (2 µg, 10% of input). Lane 4: HCMV lysate (30 µg, 1% of input). Note that spontaneous cleavage of GST occurred in the GST-SMARCB1 protein sample (Lanes 2 and 3). The asterisks (*) on Lanes 1 and 4 indicates unspecific bands by the use of anti-SMARCB1.</p

Increased expression of the SWI/SNF core subunits in HCMV infected cells.

<p>(<b>A</b>) The expression of UL114, SMARCB1 and UL44 in nuclear extracts (20 µg) from mock and HCMV-infected fibroblast cells was analyzed at immediate –early (12 hpi), early (24 hpi) and late (48 and 72 hpi) times of infection by western blot. The western blots were analyzed by Thyphoon scanning. GAPDH was used as a loading control. (<b>B</b>) The expression of BRG1, BAF155 and BAF 170 in nuclear extracts (40 µg) from mock and HCMV-infected fibroblast cells was analyzed at late (72 hpi) time of infection by western blot. The western blots were analyzed by Thyphoon scanning. GAPDH was used as a loading control.</p

Interaction of SMARCB1 and UL44 in HCMV-infected fibroblast cells and with recombinant proteins.

<p>(<b>A</b>) Co-immunoprecipitation of endogenous SMARCB1 and UL44 in HCMV-infected fibroblast cells. Equal numbers of mock infected and HCMV infected fibroblast cells were lysed at 72 hpi, and the extracts were immunoprecipitated with anti-SMARCB1. Co-immunoprecipitated proteins were resolved electrophoretically and subjected to immunoblot analysis with anti-UL44 and anti-SMARCB1. Lane 1: Mock lysate immunoprecipitated with anti-SMARCB1. Lane 2: HCMV lysate immunoprecipitated with anti-SMARCB1. Lane 3: HCMV lysate immunoprecipitated with no antibody. Lanes 4 and 5, SMARCB1 and UL44 input in extracts (7 µg, 1% of the total in Lane 4 and 35 µg, 5% of the total in Lane 5). IgGHc: IgG heavy chain. (<b>B</b>) <i>In vitro</i> pull-down assay of GST-SMARCB1 and His₆-UL44. Purified GST-SMARCB1 or GST incubated with purified His₆-UL44 immobilized on magnetic His-tag Dynabeads. Samples were analyzed by SDS-PAGE and Coomassie blue staining. Lane 1: GST-SMARCB1+Dynabeads His-tag. Lane 2: His₆-UL44+Dynabeads His-tag. Lane 3: GST-SMARCB1+His₆-UL44+Dynabeads His-tag. Lane 4: His₆-UL44 (2 µg, 10% of input). Lane 5: GST-SMARCB1 (2 µg, 10% of input). Lane 6: GST+His₆-UL44+Dynabeads His-tag. Lane 7: GST (2 µg, 10% of input). Note that spontaneous cleavage of GST occurred in the GST-SMARCB1 protein sample (Lane 5). (^……) indicates that samples were run on the same gel.</p

Recruitment of SWI/SNF chromatin remodeling factors to nuclear virus DNA replication foci.

<p>(<b>A</b>) SMARCB1 co-localizes with UL44 in HCMV-infected fibroblast cells harvested at 24, 48, and 72 hpi. (<b>B</b>) Co-localization of SMARCB1 and other essential factors of the SWI/SNF complex; BRG-1, BAF155, BAF170, in HCMV infected fibroblast cells harvested at 48 hpi. The cells were fixed and subjected to double-staining for UL44 (mouse Mab-UL44) and SMARCB1 (rabbit Pab-SMARCB1) and SMARCB1 (rabbit Pab-SMARCB1) and either BRG-1 (mouse Mab-BRG-1), BAF155 (mouse Mab BAF155), BAF170 (mouse Mab BAF170) for immunofluorescence microscopy. Secondary antibodies were used for staining UL44, BRG-1, BAF155, BAF170 in green (anti-mouse 488) and SMARCB1 in red (anti-rabbit 594), and the cells were further visualized by confocal microscopy. Co-localization was visualized by a merge of the two microscopic determinations, and counterstaining of the nuclei was achieved by the use of DAPI.</p

SMARCB1, UL114 and UL44 are associated with the chromatin and the nuclear matrix in HCMV infected fibroblast cells.

<p>Mock- and HCMV-infected fibroblast cells harvested at indicated time points were subjected to sub-nuclear fractionation to obtain whole chromatin fraction and core nuclear matrix. Proteins from equal cell equivalents from each fraction were analyzed by western blotting with the indicated antibodies.</p

Markers of inflammation in relation to CMV-disease.

<p>Plasma levels of markers of inflammation, marker of endothelial cell activation and chemokines in relation to clinical manifestation of CMV disease as either CMV syndrome or tissue invasive CMV disease. The P-values are adjusted for potential confounders (i.e., age, sex, viral load and time from transplantation). TI, tissue invasive. P-values <0.00625 were considered significant to account for multiple testing (see Methods). Data are given as mean and 95% CI.</p

Demographic data of patients suspected to have CMV disease and confirmed or not confirmed by a central analyses of CMV DNAemia.

<p>[a] chi square.</p><p>[b] t-test.</p><p>[c] Mann-Whitney test.</p

Composite score constructed from the multivariable logistic coefficients and ROC curve cut-offs of predictors of tissue invasive CMV disease and corresponding diagnostic characteristics.

<p>If ≥4 points: OR 4.7 (95% CI: 2.6–8.4) for having tissue invasive CMV disease.</p

Markers of inflammation in relation to CMV-DNAemia.

<p>Plasma levels of markers of inflammation, marker of endothelial cell activation and chemokines in relation to presence or absence of CMV DNAemia in patients suspected to have CMV disease. The P-values are adjusted for potential confounders (i.e., age, sex and time from transplantation). P-values <0.00625 were considered significant to account for multiple testing (see Methods). Data are given as mean and 95% CI.</p