Human Cytomegalovirus UL114 Interact with SMARCB1 Lane 5: UL114-c-myc+HA-antibody. Lane 6: D379-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 mg, 10% of input). Lane 4: Purified UL114 (1 mg, 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 mg, 10% of input). Lane 4: HCMV lysate (30 mg, 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. doi:10.1371/journal.pone.0034119.g002 and UL44 in nuclear protein extracts at immediate –early (12 hpi), early (24 hpi) and late (48 and 72 hpi) times of infection. Results showed that the expression of SMARCB1 increased in both mock (72 hpi) and HCMV-infected cells (Figure 4A). Also, UL114 and UL44 increased throughout the infection cycle according to previously published data (Figure 4A) [26]. Next we examined nuclear extracts at late (72 hpi) times of infection for the expression of BRG1, BAF 170 and BAF 155. As seen in Figure 4B, all three proteins showed higher expression in HCMV infected cells compared to mock cells. SMARCB1 and UL44 interact in vivo and in vitro As co-localization studies indicated that SMARCB1 was associated with the viral replication apparatus, experiments investigating the interaction between SMARCB1 and UL44 were undertaken. First, we performed experiments in which SMARCB1 was immunoprecipitated with anti-SMARCB1 antibody from cell extracts prepared from HCMV-infected fibroblast cells. Western blot analysis of the immunoprecipitated samples revealed the presence of UL44 (Figure 5A, lane 2). UL44 could not be detected in IP experiments with no antibody or from uninfected cells (mock) (Figure 5A, lanes 3 and 1, respectively). The interaction between SMARCB1 and UL44 could be mediated by UL114, other viral and cellular proteins or DNA. We thus performed an experiment with purified proteins to analyze if it was a direct interaction between SMARCB1 and UL44. UL44 was cloned into a bacterial His6 expression vector and purified. A His6 pull-down assay was carried out using purified His6-UL44 immobilized to magnetic beads with excess of purified GST-SMARCB1 and GST. His6- UL44 was able to precipitate GST-SMARCB1 but not GST alone (Figure 5B, lanes 3 and 6, respectively). The specificity of the binding reaction assay was tested using His6-NEIL1 instead of His6-UL44 as an irrelevant protein (Figure S3). We have thus demonstrated a direct interaction between UL44 and SMARCB1. Expression and sub-nuclear distribution of SMARCB1, UL114, and UL44 in the nuclei of HCMV-infected fibroblast cells Biochemical and genetic evidence suggest that the SWI/SNF complex is involved in the remodeling of chromatin during gene activation [24,29,30]. Studies have shown that several components of the SWI/SNF complex are enriched in active chromatin and are associated with the nuclear matrix [31]. We therefore examined the sub-nuclear localization of SMARCB1, UL114 and UL44 in mock and HCMV-infected fibroblasts at early (24 hpi) and late (48) times of infection by biochemical fractionation and western blot analysis. Extracts from mock-and HCMV-infected cells were prepared to obtain the soluble chromatin and the nuclear matrix and analyzed by western blotting. The fractionation procedure was controlled by immuno- blotting with antibodies directed against lamin A/C, a nuclear matrix-associated protein, and histone H1, a chromatin-associated protein. Immunoblotting (Figure 6) revealed that SMARCB1, UL114 and UL44 were present in both the soluble chromatin and nuclear matrix fractions of the HCMV-infected cells. Both mock- and HCMV-infected cells showed highest enrichment of SMARCB1 in the nuclear matrix fraction 48 hpi. The UL114 and UL44 proteins showed the highest enrichment in the chromatin fraction and the expression increased 24–48 hours after HCMV infection. Discussion Evidence from several studies indicates that the DNA genome of herpesviruses is devoid of any nucleosomes within the virus particle [32–34]. In contrast, in infected nuclei, herpesvirus genomes form structures that resemble cellular chromatin and these structures change in composition throughout the time course of infection [35–38]. As early as two hours post infection, a fraction of the HCMV genomes is associated with histones, but eventually, the HCMV progeny genomes have to be stripped naked before being packaged [9]. Factors involved in DNA replication, repair and transcription do not get access to DNA packed in chromatin and thus have to act in concert with chromatin modifiers and remodelers that loosen the chromatin grip on DNA. The SWI/SNF family of chromatin remodeling complex utilizes the energy of ATP hydrolysis to remodel chromatin structures, thereby participating in gene regulation, replication, viral integration, control of cell growth and tumor suppression [25,39]. SMARCB1 was initially identified as a cellular partner to the HIV-1 integrase [40]. Subsequent studies have revealed the interaction of SMARCB1 or other subunits of the SWI/SNF complex with viral proteins from human papillo- mavirus [41–43], Epstein-Barr virus [44], Kaposi’s sarcoma- associated herpes virus [45] and herpes simplex virus 21 [46,47]. Collectively, these studies have shown that the SWI/SNF complex is crucial for effective viral gene transcription and DNA replication. In search for cellular partners of UL114, a nearly full-length SMARCB1 clone was identified as an interacting partner. The direct interaction between UL114 and SMARCB1 was validated in vitro by three different experiments. UL114 has been found to interact with UL44 and this complex was highly enriched in viral replication foci [16,26]. Since co- localization between UL114 and SMARCB1 could not be carried out, UL44 was used as a replication marker. By this mean we showed that SMARCB1 co-localizes to replication foci as early as 24 hpi. The SWI/SNF complex consists of at least nine subunits that are conserved among eukaryotes [48]. Four of the subunits; the central ATPase subunit, either hBRM (Bramha) or Bramha- related gene-1 (BRG-1), SMARCB1, BAF170 and BAF155 are required for efficient chromatin remodeling [28]. Our results demonstrate that the expression of these core subunits of the SWI/ SNF chromatin remodeling complex increased during HCMV infection and were relocated to and concentrated in virus replication foci of HCMV-infected fibroblasts. To our knowledge, PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e34119