The assembly and organization of nucleosomes on viral genomes is thought to be linked to the replication mechanisms and life cycle of the virus [17]. Modifications of the chromatin structure on HCMV-DNA are thus of importance both for transcription, replication and regulation of latency [18–21]. The chromatin structure on DNA is modified by post-translational modification, e.g. acetylation and methylation of N-terminal tails of histone proteins and by chromatin remodeling [22–24]. There are currently four different families of chromatin remodeling complexes; SWI/SNF (switching defective/sucrose non-ferment- ing), ISWI (imitation switch), CDH (chromo domain helicase DNA-binding) and INO80 (inositol requiring 80) family [23,25]. These ATP dependent chromatin remodeling complexes work in concert with histone tail modifying enzymes [24]. In order to elucidate the mechanisms of UL114 mediated enhancement of HCMV-DNA-replication we have searched for cellular partners to UL114 by a two-hybrid assay. One of the clones identified by sequencing was the human SMARCB1 protein, a core subunit of the highly conserved multi subunit SWI/SNF chromatin remodeling complex. In this work we characterized the intracellular localization of SMARCB1 through- out infection showing re-localization to the replication foci. Further experiments showed direct interaction of SMARCB1 and UL114 and SMARCB1 and UL44. Results Yeast two-hybrid experiments identify the cellular factor SMARCB1 as a strong binding partner to UL114 To identify cellular partners of the HCMV encoded uracil DNA glycosylase, UL114, we used a yeast based two-hybrid system with a ‘‘bait’’ plasmid (pGBKT7-UL114) encoding the full-length UL114 protein fused to GAL4 DNA-binding domain (BD) in pGBKT7 transformed into the yeast strain AH109. The two-hybrid screen was performed by mating a yeast strain Y187 pre-transformed with a highly complex brain cDNA library cloned into the yeast GAL4 activation domain (AD) vector pACT2 with AH109-pGBKT7- UL114. Cells were grown on synthetic dropout (SD) medium; SD- trp/-leu/-his/-ade, to select for strong interactions. More than 350 clones were found to grow under these strict conditions indicating several putative cellular partners to UL114. All of these clones were subjected to automated sequencing and homology searches in NCBI databases. Because a large number of clones were screened, several independent clones containing the same binding domain were isolated in many cases. After sorting the sequencing data eliminating typically false positives and taking into account clones represented more than once, 17 clones were subjected to direct two- hybrid analysis (Figure 1A). Self-activating clones were identified by mating the 17 clones with the empty BD vector (pGBKT7) and plating in selective media: 2leu/2trp/+his/+ade for cell viability and 2leu/2trp/2his/2ade for self-activation (clones 2, 3, 5, 7, 8, 9, 10, 13 and 14; Figure 1A). One of the clones that did not self- activate (clone 12, Figure 1A) was identified as a partial cDNA encoding for human SMARCB1 (SNF5/INI1/BAF47) (379 aa from accessory number NM_003073.2, Gene ID: 6598), termed D379-SMARCB1 in this paper (Figure 1B). The specific interaction between D379-SMARCB1 and UL114 was confirmed by direct two-hybrid analysis with various controls (Figure 1C). SMARCB1 and UL114 interact in vitro In order to test whether the interaction between UL114 and D379-SMARCB1 was reproducible in vitro, three sets of experi- ments were performed. First, by coupled in vitro transcription/ translation and co-immunoprecipitation (IP), we examined the Human Cytomegalovirus UL114 Interact with SMARCB1 interaction between the two proteins using [35S]methionine- labeled HA-tagged D379-SMARCB1 and [35S]methionine-la- beled c-myc-tagged UL114. IP with anti-HA antibody recovered HA-D379-SMARCB1 and c-myc-UL114 (Figure 2A, lane 3). Reciprocally, c-myc-UL114 and HA-D379-SMARCB1 were detected when immunoprecipitation was performed with anti-c- myc antibody (Figure 2A, lane 4). Control immunoprecipitations with an irrelevant antibody and protein were carried out to confirm the specificity of the interaction between D379- SMARCB1 and UL114. Thus, c-myc-tagged UL114 was not immunoprecipitated by the HA antibody (Figure 2A, lane 5) and HA-tagged D379-SMARCB1 was not immunoprecipitated by the c-myc antibody (Figure 2A, lane 6). Moreover, an independent HA-tagged protein HA-clone 4 (clone 4 from Figure 1A) was not immunoprecipitated by c-myc-UL114 (Figure S1). Second, direct interaction studies between UL114 and SMARCB1 were carried out using a glutathione S-transferase (GST) pull-down assay. To this end, full-length SMARCB1 was cloned into a bacterial GST expression vector and purified. GST was also expressed; however the expression was so high that no purification was needed for these experiments. GST-SMARCB1 or crude GST extract incubated with purified UL114 were subjected to conventional pull-down analysis by the use of glutathione (GST-binding) magnetic beads. After pull-down, the samples were analyzed by western blotting using anti-UL114, anti- SMARCB1 and anti-GST antibodies. Western blot results shown in Figure 2B, indicated that UL114 bound to SMARCB1 directly. Third, to investigate whether SMARCB1 and UL114 interacted in vivo, human fibroblast cells were infected with HCMV. The lysates were immunoprecipitated with anti-SMARCB1 antibodies. Immunoprecipitation with SMARCB1 antibody clearly showed input SMARCB1, however, we could not detect UL114 in the precipitate (data not shown). As previously reported the reverse co- immunoprecipitation experiment with UL114 antibodies was not possible because the available UL114 antibody lacked immuno- precipitation properties [26]. Thus, GST-SMARCB1 and GST immobilized on GST-binding magnetic beads were incubated with HCMV-infected cell lysates. After pull-down, the samples were analyzed by western blotting using anti-UL114, anti-SMARCB1, anti-GST and anti-UL57. As shown in Figure 2C, UL114 from HCMV infected cells was found to interact robustly with GST- SMARCB1 but not to GST alone. The specificity of the assay was tested by blotting against UL57, the single-stranded DNA binding protein. As seen in Figure 2C, UL57 was readily detected in the HCMV lysate (lane 4), however, it was not precipitated with GST- SMARCB1. These in vitro binding results confirm the yeast two- hybrid interaction between UL114 and SMARCB1. SMARCB1 is recruited to HCMV replication foci As described previously the essential viral replication proteins organize into discrete nuclear foci, termed pre-replication sites that mature into viral DNA replication compartments [26,27]. UL44 is recruited into pre-replication sites already at 5 hours post infection (hpi), prior to the onset of DNA replication and can thus be used as a HCMV replication focus marker [16,26]. We investigated whether SMARCB1 was recruited to these sites during HCMV infection by assessing intracellular localization of SMARCB1 in mock and HCMV-infected fibroblast cells at immediate –early (5– 12 hpi), early (24 hpi) and late (48 and 72 hpi) time points of infection. We showed that SMARCB1 was recruited to viral replication foci in HCMV-infected cells and co-localized with UL44 throughout the infection cycle (24–72 hpi) (Figure 3A). In mock fibroblast cells the localization of SMARCB1 was hampered by high background staining in contrast to HCMV infected PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e34119