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. 2009 Sep 15;392(1):16-23.
doi: 10.1016/j.virol.2009.06.035. Epub 2009 Jul 23.

Interaction of HCMV UL84 with C/EBPalpha transcription factor binding sites within oriLyt is essential for lytic DNA replication

Dominique Kagele  1 , Yang Gao, Kate Smallenburg, Gregory S Pari
Affiliations

Interaction of HCMV UL84 with C/EBPalpha transcription factor binding sites within oriLyt is essential for lytic DNA replication

Dominique Kagele et al. Virology. .

Abstract

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.

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Figures

Figure 1
Figure 1. Interaction of UL84, UL44, IE2 and C/EBPα with oriLyt
(A) Schematic of HCMV genome showing the location of oriLyt and the positions of three essential regions, various restriction enzyme recognition sites, RNA/DNA hybrids, C/EBPα/β, IE2-binding sites, oriLyt promoter region and the RNA stem-loop structure. Also shown are the relative locations of PCR primers used for ChIP assays for DNA isolated from infected cells and purified virions. (B) ChIP assays showing the interaction of UL84, IE2, UL44 and C/EBPα with various regions of oriLyt. Infected cells were prepared as described and specific antibodies to UL84 (Virusys), IE2 (Vancouver Biotech), UL44 (Bill Britt) or C/EBPα (Santa Cruz) were used for immunoprecipitations. PCR primers from regions 1-5 are shown above each ChIP assay gel. The lanes of each ChIP assay are as follows: 1, PCR product from input DNA; 2, PCR product from immunoprecipitations using specific antibodies shown to the left of the figure; 3, PCR product from immunoprecipitations using an unrelated antibody that was the same isotype as the test antibody. Also shown is a PCR amplification of the HCMV UL144 loci from samples immunoprecipitated using the anti-UL84 antibody. Lanes: 1, PCR product from input DNA; 2, PCR product from immunoprecipitations using the anti-UL84 antibody; 3, PCR product from immunoprecipitations using an unrelated antibody that was the same isotype as the test antibody. Solid line shows sequences gaps.
Figure 2
Figure 2. C/EBPα interacts with the proposed oriLyt C/EBPα-binding site sequence in vitro
Nuclear extract was prepared from HEK293 cells transfected with a C/EBPα expression plasmid. Three microliters of nuclear extract was incubated with ds oligonucleotides in the presence or absence of a C/EBPα specific antibody. Lanes: 1, control oligonucleotide; 2, control oligonucleotide plus nuclear extract from cells transfected with a C/EBPα expression plasmid; 3, control oligonucleotide plus nuclear extract from cells transfected with a C/EBPα expression plasmid plus a C/EBPα specific antibody; 4, control oligonucleotide plus nuclear extract from cells transfected with a C/EBPα expression plasmid plus 20X; unlabeled control oligonucleotide; 5, control oligonucleotide plus untrasfected nuclear extract; 6; oriLyt a C/EBPα oligonucleotide; 7, orilyt C/EBPα oligonucleotide plus nuclear extract from cells transfected with a C/EBPα expression plasmid; 8, oriLyt C/EBPα oligonucleotide plus nuclear extract from cells transfected with a C/EBPα expression plasmid plus a C/EBPα specific antibody; 9, oriLyt C/EBPα oligonucleotide plus nuclear extract from cells transfected with a C/EBPα expression plasmid plus 20X; unlabeled control oligonucleotide; 10, oriLyt C/EBPα oligonucleotide plus untrasfected nuclear extract; 6; oriLyt a C/EBPα oligonucleotide.
Figure 3
Figure 3. Intact C/EBPα sites are required for amplification of oriLyt
(A) Schematic of oriLyt sequence from nucleotides 92,174-92,541 showing the positions of C/EBPα transcription factor binding sites and the mutations made within the oriLyt sequence. (B) Southern blot of a transient transfection replication assay. HF cells were transfected with various oriLyt containing plasmids plus the internal control plasmid pALTER-OriLyt. Cells were infected 24 h post transfection and total cell DNA was harvested 5 days post transfection and cleaved with DpnI and EcoRI. Following agarose gel separation and transfer to a nylon membrane, the blot was hybridized to a 32P-labbled-pGEM probe. Arrows to the left of the figure show the identification of each plasmid.
Figure 4
Figure 4. C/EBPα does not interact with UL84 in infected or transfected cells
HF cells were infected with HCMV and protein lysates were prepared 5 days post infection. Proteins were immunoprecipitated using either a UL84 or C/EBPα - specific antibody. Immunoprecipitated protein was analyzed by western blot using the same antibodies. Lanes: 1, infected cell lysate, 2, infected cell protein lysate immunoprecipitated with anti- C/EBPα 3, Infected cell protein lysate; 4, infected cell protein lysate immunoprecipitated with anti- UL84; 5, UL84 and C/EBPα cotransfected cell protein lysate; 6, cotransfected protein lysate immunoprecipitated with anti-UL84; 7, UL84 and C/EBPα cotransfected cell protein lysate; 8, cotransfected protein lysate immunoprecipitated with anti-C/EBPα. The specific antibodies used to react with the Western blot are shown to the left of the figure.
Figure 5
Figure 5. Mutation of C/EBPα site 2 results in the loss of UL84 binding in transfected cells
HF cells (1 × 107) were transfected by electroporation with either wt OriLyt (10μg) or pOriLyt-2/3 (10μg) and a UL84 (5μg) expression plasmid. Two days post transfection cells were prepared for ChIP assays using a UL84 specific antibody or, in the case of the control ChIP an unrelated antibody of the same isotype as the UL84 antibody. Lanes: 1, PCR product input DNA from cells cotransfected with wt OriLyt containing plasmid and a UL84 expression plasmid; 2, PCR product from a ChIP assay from cells transfected with an OriLyt containing plasmid and a UL84 expression plasmid; 3, PCR product from a ChIP assay from cells transfected with an OriLyt containing plasmid and a UL84 expression plasmid using an isotype control antibody. PCR reactions were performed using primers that flanked the C/EBPαβ transcription factor binding sites with in oriLyt: Forward 5′-ACTCGAGTCACCATCCCATAAT-3′ and reverse 5′-TTTTCGTAGAACGTTTCGTTAGAAG-3′. The plasmid used in the transfection experiments is shown at the left of the figure.
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