Human Cytomegalovirus DNA Replication Requires Transcriptional Activation via an IE2- and UL84-Responsive Bidirectional Promoter Element within ori Lyt
Open Access
- 1 November 2004
- journal article
- Published by American Society for Microbiology in Journal of Virology
- Vol. 78 (21) , 11664-11677
- https://doi.org/10.1128/jvi.78.21.11664-11677.2004
Abstract
Amplification of the human cytomegalovirus (HCMV) lytic origin ( ori Lyt) in human fibroblasts is dependent upon six core replication proteins and UL84, IE2, and UL36-38. Using a telomerase-immortalized human fibroblast cell line (T-HFs), ori Lyt-dependent DNA replication no longer required the gene products of UL36-38. To determine the role of IE2 in DNA replication in human fibroblasts, we examined potential IE2-binding sites within HCMV ori Lyt. We now show that a strong bidirectional promoter ( ori Lyt PM ) (nucleotides 91754 to 92030) is located in the previously identified core region of the origin and is required for efficient amplification of ori Lyt. It was determined that a 14-bp novel DNA motif ( ori Lyt promoter activation element), which was initially identified as a binding element for the immediate-early protein IE2, was essential for ori Lyt PM activity. In Vero cells the ori Lyt PM was constitutively active and strongly repressed by IE2, but it was reactivated by UL84. In contrast, transfection of the ori Lyt PM into human fibroblasts resulted in a very low basal level of promoter activity that was dramatically up-regulated upon infection with HCMV. Cotransfection assays demonstrated that the transfection of UL84 along with IE2 transactivated the ori Lyt PM in human fibroblasts. Further activation was observed upon cotransfection of the set of plasmids expressing the entire replication complex. Efficient ori Lyt amplification in the absence of IE2 in human fibroblasts was observed by replacing the ori Lyt PM with the simian virus 40 early promoter. Under these conditions, however, UL84 was still required for amplification of ori Lyt. These results suggest that the mechanism of initiation of HCMV lytic replication in part involves transcriptional activation.Keywords
This publication has 47 references indexed in Scilit:
- Genomic structure of the human mitochondrial chaperonin genes: HSP60 and HSP10 are localised head to head on chromosome 2 separated by a bidirectional promoterHuman Genetics, 2003
- Human Cytomegalovirus UL84 Localizes to the Cell Nucleus via a Nuclear Localization Signal and Is a Component of Viral Replication CompartmentsJournal of Virology, 2002
- Identification of additional IE2-p86-responsivecis-repressive sequences within the human cytomegalovirus major immediate early gene promoterJournal of Biomedical Science, 2002
- Identification of Additional IE2-p86-Responsive cis-Repressive Sequences within the Human Cytomegalovirus Major Immediate Early Gene PromoterJournal of Biomedical Science, 2002
- Identification of a Cellular Protein That Interacts and Synergizes with the RTA (ORF50) Protein of Kaposi's Sarcoma-Associated Herpesvirus in Transcriptional ActivationJournal of Virology, 2001
- Sequence Requirements for Interaction of Human Herpesvirus 7 Origin Binding Protein with the Origin of Lytic ReplicationJournal of Virology, 2001
- Identification of Functional Elements in the Bidirectional Promoter of the Mouse Nthl1 and Tsc2 GenesBiochemical and Biophysical Research Communications, 2000
- Comparison of DNA Sequences with Protein SequencesGenomics, 1997
- Human cytomegalovirus late protein encoded by ie2: a trans-activator as well as a repressor of gene expressionJournal of General Virology, 1994
- Two binding sites for the herpes simplex virus type 1 UL9 protein are required for efficient activity of the oriS replication originJournal of General Virology, 1990