(A) In vitro 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 in vitro 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) and (C) GST pull-down assays to detect the interaction of SMARCB1 and UL114. (B) Purified GST-SMARCB1 or crude extract of E. coli 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). (C) Purified GST-SMARCB1 or crude extract of E. coli 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.

(A) 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) 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.

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.

Two-hybrid screening of a brain cDNA library (activation domain (AD)) using UL114 as a bait (binding domain (BD)) identified several potential interacting cellular partners. (A) 17 unique clones were subjected to direct two-hybrid analysis. Self-activation of the reporter genes lacZ and HIS3 was tested for each of the 17 clones by co-transforming and plating each of the putative interacting clones (interactors 1 to 17-AD) and the empty bait plasmid (empty-BD) on selective media: (−leu/−trp/+his/+ade) for cell viability and (−leu/−trp/−his/−ade) for self-activation. Single colonies diluted in water at equal density were spotted onto selective media as indicated. Clones 1–11 and 13–17: interactor 1–11 and 13–17+BD-empty (pGBKT7); clone 12: AD-Δ379-SMARCB1+BD-empty (pGBKT7); clone 18: positive control, AD-Tag+BD-p53. Clones 1, 4, 6, 11 and 12 did not self-activate. (B) Schematic presentation of wild type (wt) SMARCB1 and truncated Δ379-SMARCB1 identified by the two-hybrid analysis (clone 12 from Figure 1A). Numbers indicate amino-acid residues. SMARCB1 has two highly conserved domains (Rpt1 and Rpt2) that are imperfect direct repeats of each other and a third conserved coiled coil domain at the C terminus (CC). (C) Extensive direct two-hybrid analysis of truncated SMARCB1 (Δ379-SMARCB1). Strong interaction was tested by co-transformation and plating of AD-Δ379-SMARCB1+BD-UL114 on selective media (−leu/−trp/−his/−ade). Self-activation was tested by co-transformation and plating of AD-Δ379-SMARCB1+BD-empty and AD-empty+BD-UL114 on selective media (−leu/−trp/−his/−ade). Four single colonies diluted into water at equal density were spotted onto selective media as indicated. Positive control: AD-Tag+BD-p53, Negative control: AD-empty+BD-empty.

(A) SMARCB1 co-localizes with UL44 in HCMV-infected fibroblast cells harvested at 24, 48, and 72 hpi. (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.

(A) 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) In vitro 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.