title: An Epstein-Barr Virus-Encoded Protein Complex Requires an Origin of Lytic Replication In Cis to Mediate Late Gene Transcription.
abstract: Epstein-Barr virus lytic replication is accomplished by an intricate cascade of gene expression that integrates viral DNA replication and structural protein synthesis. Most genes encoding structural proteins exhibit "true" late kinetics-their expression is strictly dependent on lytic DNA replication. Recently, the EBV BcRF1 gene was reported to encode a TATA box binding protein homolog, which preferentially recognizes the TATT sequence found in true late gene promoters. BcRF1 is one of seven EBV genes with homologs found in other β- and γ-, but not in α-herpesviruses. Using EBV BACmids, we systematically disrupted each of these "βγ" genes. We found that six of them, including BcRF1, exhibited an identical phenotype: intact viral DNA replication with loss of late gene expression. The proteins encoded by these six genes have been found by other investigators to form a viral protein complex that is essential for activation of TATT-containing reporters in EBV-negative 293 cells. Unexpectedly, in EBV infected 293 cells, we found that TATT reporter activation was weak and non-specific unless an EBV origin of lytic replication (OriLyt) was present in cis. Using two different replication-defective EBV genomes, we demonstrated that OriLyt-mediated DNA replication is required in cis for TATT reporter activation and for late gene expression from the EBV genome. We further demonstrate by fluorescence in situ hybridization that the late BcLF1 mRNA localizes to EBV DNA replication factories. These findings support a model in which EBV true late genes are only transcribed from newly replicated viral genomes.
authors.affiliation: Department of Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.|Department of Oncology (McArdle Laboratory for Cancer Research), University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.
authors.forename: Reza
authors.initials: R
authors.lastname: Djavadian
authors.affiliation: Department of Oncology (McArdle Laboratory for Cancer Research), University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.|Research Center for Emerging Viral Infections, Chang Gung University, Taoyuan, Taiwan.|Department of Microbiology and Immunology, Chang Gung University, Taoyuan, Taiwan.|Department of Medical Laboratory, Chang Gung Memorial Hospital, Linkou, Taiwan.
authors.forename: Ya-Fang
authors.initials: YF
authors.lastname: Chiu
authors.affiliation: Department of Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.|Department of Oncology (McArdle Laboratory for Cancer Research), University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.
authors.forename: Eric
authors.initials: E
authors.lastname: Johannsen
mesh_terms: D004261:DNA Replication / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D005814:Genes, Viral / Q000235:genetics; D057809:HEK293 Cells; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D017404:In Situ Hybridization, Fluorescence; D016133:Polymerase Chain Reaction; D018741:Replication Origin / Q000235:genetics*; D014158:Transcription, Genetic; D014779:Virus Replication / Q000235:genetics*
chemical_list: D004279:DNA, Viral