Interaction of HCMV UL84 with C/EBPalpha transcription factor binding sites within oriLyt is essential for lytic DNA replication.
Human cytomegalovirus (HCMV) lytic DNA replication is initiated at the cis-acting oriLyt region and requires six core replication proteins along with UL84 and IE2. Although UL84 is thought to be the replication initiator protein, little is known about its interaction with oriLyt. We have now performed chromatin immunoprecipitation assays (ChIP) using antibodies specific to UL84, IE2, UL44, CCAAT/enhancer binding protein (C/EBPalpha) and PCR primers that span the entire oriLyt region to reveal an evaluation of specific protein binding across oriLyt. UL84 interacted with several regions of oriLyt that contain C/EBPalpha transcription factor binding sites. Mutation of either of one of C/EBPalpha (92,526 or 92,535) sites inactivated oriLyt and resulted in the loss of binding of UL84. These data reveal the regions of interaction within oriLyt for several key replication proteins and show that the interaction between UL84 and C/EBPalpha sites within oriLyt is essential for lytic DNA replication.
Kagele
Dominique
D
University of Nevada-Reno School of Medicine, Department of Microbiology and Immunology and the Cell and Molecular Biology Graduate Program, Howard Bldg. 210, Reno, NV 89557, USA.
Gao
Yang
Y
Smallenburg
Kate
K
Pari
Gregory S
GS
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics* / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D020221:Optic Nerve Injuries; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D022763:CCAAT-Enhancer-Binding Protein-alpha; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C072217:ICP36 protein, Cytomegalovirus; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D014764:Viral Proteins; C112091:oriLyt protein, Human cytomegalovirus; C072218:UL84 protein, Cytomegalovirus