Human Cytomegalovirus UL114 Interact with SMARCB1 Figure 1. Direct yeast two-hybrid analysis of putative interacting partners of UL114. 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: (2leu/2trp/+his/ +ade) for cell viability and (2leu/2trp/2his/2ade) 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-D379-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 D379-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 (D379-SMARCB1). Strong interaction was tested by co-transformation and plating of AD- D379-SMARCB1+BD-UL114 on selective media (2leu/2trp/2his/2ade). Self-activation was tested by co-transformation and plating of AD-D379- SMARCB1+BD-empty and AD-empty+BD-UL114 on selective media (2leu/2trp/2his/2ade). 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. doi:10.1371/journal.pone.0034119.g001 fibroblast cells (Figure S2 and Figure 3B). Likewise, attempts to visualize co-localization at immediate –early (5–12 hpi) time points of infection were hampered by high background staining (data not shown). Since only SMARCB1 and UL114 rabbit antibodies functioning in immunofluorescence were available, co-localization studies could not be carried out. However, previous results have shown that UL114 localizes to viral replication foci as early as 5 hpi and co-localizes with UL44 throughout the infection cycle (5–72 hpi) [26], thus indirectly indicating that SMARCB1 and UL114 co-localize. To investigate whether the entire 2 MDa multi SWI/SNF complex or only the SMARCB1 protein was recruited to the viral replication foci, we examined co-localization between UL44 and three other proteins in the multi SWI/SNF complex; the central ATPase subunit; Bramha-related gene-1 (BRG-1), BAF170 and BAF155, which in addition to SMARCB1 are required for efficient chromatin remodeling activity [28]. As shown in Figure 3B they were all recruited into HCMV replication foci (48 hpi). The expression of the core SWI/SNF chromatin remodeling factors increase in HCMV-infected fibroblast cells The experiments shown in Figure 3 suggested that the expression of SWI/SNF members tested increased during HCMV infection. We first, analyzed the expression of SMARCB1, UL114 PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e34119