Interaction of the human cytomegalovirus uracil DNA glycosylase UL114 with the viral DNA polymerase catalytic subunit UL54. Strang Blair L,Coen Donald M The Journal of general virology Interaction between human cytomegalovirus uracil DNA glycosylase (UL114) and the viral DNA polymerase accessory subunit (UL44) has been reported; however, no such association was found in proteomic studies of UL44-interacting proteins. Utilizing virus expressing FLAG-tagged UL114, nuclease-resistant association of UL44 and the DNA polymerase catalytic subunit UL54 with UL114 was observed by co-immunoprecipitation. Contrary to a previous report, we observed that UL114 was much less abundant than UL44. Interaction of UL114 with UL54, independent of the UL54 carboxyl terminus, but not with UL44 was detected in vitro. Our data are consistent with a direct UL114-UL54 interaction, and suggest that UL114 and UL54 act in concert during base excision repair of the viral genome. 10.1099/vir.0.022160-0

The chromatin remodeling factor SMARCB1 forms a complex with human cytomegalovirus proteins UL114 and UL44. PloS one 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 co-immunoprecipitation 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 in different DNA transactions. 10.1371/journal.pone.0034119

Characterization of human cytomegalovirus uracil DNA glycosylase (UL114) and its interaction with polymerase processivity factor (UL44). Ranneberg-Nilsen Toril,Dale Hege Avsnes,Luna Luisa,Slettebakk Ragnhild,Sundheim Ottar,Rollag Halvor,Bjørås Magnar Journal of molecular biology Here, we report the molecular characterization of the human cytomegalovirus uracil DNA glycosylase (UNG) UL114. Purified UL114 was shown to be a DNA glycosylase, which removes uracil from double-stranded and single-stranded DNA. However, kinetic analysis has shown that viral UNG removed uracil more slowly compared with the core form of human UNG (Delta84hUNG), which has a catalytic efficiency (k(cat)/K(M)) 350- to 650-fold higher than that of UL114. Furthermore, UL114 showed a maximum level of DNA glycosylase activity at equimolar concentrations of the viral polymerase processivity factor UL44. Next, UL114 was coprecipitated with DNA immobilized to magnetic beads only in the presence of UL44, suggesting that UL44 facilitated the loading of UL114 on DNA. Moreover, mutant analysis demonstrated that the C-terminal part of UL44 (residues 291-433) is important for the interplay with UL114. Immunofluorescence microscopy revealed that UL44 and UL114 colocalized in numerous small punctuate foci at the immediate-early (5 and 8 hpi) phases of infection and that these foci grew in size throughout the infection. Furthermore, coimmunoprecipitation assays with cellular extracts of infected cells confirmed that UL44 associated with UL114. Finally, the nuclear concentration of UL114 was estimated to be 5- to 10-fold higher than that of UL44 in infected cells, which indicated a UL44-independent role of UL114. In summary, our data have demonstrated a catalytically inefficient viral UNG that was highly enriched in viral replication foci, thus supporting an important role of UL114 in replication rather than repair of the viral genome. 10.1016/j.jmb.2008.05.028