RESUMEN
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.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Citomegalovirus/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Uracil-ADN Glicosidasa/química , Proteínas Virales/química , Línea Celular , Núcleo Celular/metabolismo , Cromatina/química , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/química , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Glutatión Transferasa/metabolismo , Humanos , Microscopía Fluorescente/métodos , Matriz Nuclear/metabolismo , Unión Proteica , Proteína SMARCB1 , Factores de Transcripción/química , Técnicas del Sistema de Dos Híbridos , Uracil-ADN Glicosidasa/metabolismo , Proteínas Virales/metabolismoRESUMEN
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.
Asunto(s)
Citomegalovirus/enzimología , Proteínas de Unión al ADN/metabolismo , Uracil-ADN Glicosidasa/metabolismo , Proteínas Virales/metabolismo , Sitios de Unión/genética , Células Cultivadas , Citomegalovirus/genética , Citomegalovirus/crecimiento & desarrollo , ADN/metabolismo , Proteínas de Unión al ADN/genética , Electroforesis en Gel de Poliacrilamida , Fibroblastos/citología , Fibroblastos/virología , Humanos , Inmunoprecipitación , Cinética , Microscopía Fluorescente , Mutagénesis Sitio-Dirigida , Mutación , Sistemas de Lectura Abierta/genética , Unión Proteica , Uracilo/metabolismo , Uracil-ADN Glicosidasa/genética , Proteínas Virales/genéticaRESUMEN
Regulation of DNA repair mechanisms during the viral replication cycle may have consequences for the virus with regards to genomic variability, adaptation, and replication of viral DNA. We have studied the activities and expression patterns of key enzymes involved in the first two steps of base excision repair (BER) of DNA in primary fibroblasts infected by human cytomegalovirus (HCMV). Infected cells were very proficient for removal of uracil and 5' hydrolysis of AP sites (AP endonuclease activity) as compared to the mock-infected cells, suggesting a direct role in generating free ends at uracil lesions in DNA for initiation of viral replication. Furthermore, the capacity to initiate repair of alkylated and oxidized base lesions were reduced in HCMV-infected cells, indicating increased mutation frequencies that could promote genetic variability. We hypothesize that modulation of BER activities may play an important role in HCMV pathogenesis to ensure efficient replication and genomic variation of viral DNA.