The Chromatin Remodeling Factor SMARCB1 Forms a Complex with Human Cytomegalovirus Proteins UL114 and UL44

Background: Human cytomegalovirus (HCMV) uracil DNA glycosylase, UL114, is required for efficient viral DNA replication. Presumably, UL114 functions as a structural partner to other factors of the DNA-replication machinery and not as a DNA repair protein. UL114 binds UL44 (HCMV processivity factor) and UL54 (HCMV-DNA-polymerase). In the present study we have searched for cellular partners of UL114. Methodology/Principal Findings: In a yeast two-hybrid screen SMARCB1, a factor of the SWI/SNF chromatin remodeling complex, was found to be an interacting partner of UL114. This interaction was confirmed in vitro by coimmunoprecipitation and pull-down. Immunofluorescence microscopy revealed that SMARCB1 along with BRG-1, BAF170 and BAF155, which are the core SWI/SNF components required for efficient chromatin remodeling, were present in virus replication foci 24–48 hours post infection (hpi). Furthermore a direct interaction was also demonstrated for SMARCB1 and UL44. Conclusions/Significance: The core SWI/SNF factors required for efficient chromatin remodeling are present in the HCMV replication foci throughout infection. The proteins UL44 and UL114 interact with SMARCB1 and may participate in the recruitment of the SWI/SNF complex to the chromatinized virus DNA. Thus, the presence of the SWI/SNF chromatin remodeling complex in replication foci and its association with UL114 and with UL44 might imply its involvement i

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

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

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