Figure 3. Recruitment of SWI/SNF chromatin remodeling factors to nuclear virus DNA replication foci. (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, Human Cytomegalovirus UL114 Interact with SMARCB1 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. doi:10.1371/journal.pone.0034119.g003 this is the first time a chromatin remodeling complex has been detected in virus replication foci in HCMV. Furthermore, interaction between SMARCB1 and UL44 was demonstrated both by detection of SMARCB1 in cell extracts precipitated with anti-UL44 antibodies and by direct interaction between recom- binant proteins. Thus, we have evidence of direct interactions between SMARCB1 and UL114 and SMARCB1 and UL44. The UL44 and/or UL114 proteins may be of importance to recruit and stabilize the SWI/SNF chromatin remodeling complex to the replication centers. The human HCMV DNA polymerase is composed of a catalytic subunit, UL54, which possesses basal DNA polymerase activity [49], and the accessory protein, UL44 which has been shown to specifically interact with UL54 and to stimulate long- chain DNA synthesis by UL54 [50,51]. UL44 is a multifunctional protein capable of associating/interacting with several other viral and host proteins [16,26,52–55]. Viral replication centers also serve as foci for viral gene expression, presumably in part by concentrating templates for transcription with the proteins that carry out or regulate this process. Thus, the presence of the SWI/ SNF chromatin remodeling complex in replication foci throughout infection and its association with UL114 and with UL44 might imply its involvement in different DNA transactions. For example, it has been shown that the UL44 gene product from the late viral transcript is required for efficient viral gene expression rather than viral DNA synthesis [56]. Comparable to the herpes simplex virus type-1 single-strand DNA-binding protein, ICP8, which co- precipitates with chromatin remodeling factors [47], UL44 could recruit the SWI/SNF complex to late viral promoters at late times after infection. Finally, although controversial and ill-defined, the nuclear matrix, also referred to as nucleoskeleton or scaffold, organizes the eukaryotic DNA into topologically distinct loops. This is generated by the attachment of chromatin fibers to the nuclear matrix via specific regions called scaffold or matrix attachment regions S/ MAR [57,58]. DNA replication and transcription of active DNA are found tightly associated with the nuclear matrix, while inactive loci are not [59–61]. Several studies have reported replication and expression of viral genomes in association with the nuclear matrix [62–64]. SMARCB1 has been found to be associated with the nuclear matrix and chromatin [31]. UL44 has also been shown to be associated with the nuclear matrix [65]. By fractionating the nucleus into the sub-nuclear structures; chromatin and nuclear matrix, we showed that in addition to SMARCB1 and UL44, UL114 was present in the chromatin and nuclear matrix fraction, but highly enriched in the chromatin fraction. However, it remains to be investigated whether SMARCB1, UL114 and UL44 associate in one complex and/or as different complexes (in the nuclear matrix and/or associated with chromatin) throughout the time course of infection allowing chromatin remodeling both during DNA replication, DNA transcription and DNA packaging. Materials and Methods Cells and Virus Human embryonic fibroblast (HE) cells were obtained from the National Institute of Public Health, Oslo, Norway. HE-cells were grown and maintained in 1:1 minimal essential medium PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e34119