The human cytomegalovirus origin of DNA replication (oriLyt) is the critical cis-acting sequence regulating replication-dependent late induction of the viral 1.2-kilobase RNA promoter.
Plasmid constructs containing the 1.2-kb RNA promoter from the long terminal repeat region of human cytomegalovirus (HCMV) display the early-phase regulation of this promoter but lack the characteristic late induction (E. J. Wade, K. M. Klucher, and D. H. Spector, J. Virol. 66:2407-2417, 1992). To determine if the HCMV origin of replication (oriLyt) was necessary and sufficient for the late induction of the 1.2-kb RNA promoter, we cloned a 9.6-kbp segment of the origin of replication onto the p456 OCAT plasmid containing the 1.2-kb RNA promoter. This plasmid was designated ori456 OCAT. A control construct, which contains all of the same sequences as the ori456 OCAT construct except that a 2.4-kbp segment derived from HCMV EcoRI segment U is inverted in orientation to disrupt the origin function, was designated inv456 OCAT. After electroporation into human fibroblast cells and infection with HCMV 24 h later, ori456 OCAT replicated and showed the same early and late transcription pattern as the authentic viral 1.2-kb RNA. Under similar conditions, the inv456 OCAT neither replicated nor showed late induction. Experiments using plasmids synthesized in bacteria lacking methylation activity demonstrated that the late induction was not dependent on the change in methylation state of the plasmids. Ganciclovir, an inhibitor of the HCMV DNA polymerase, was used to demonstrate the replication dependence of the expression of the virally encoded 1.2-kb RNA, while the nearby early 2.7-kb RNA was unaffected. Ganciclovir also inhibited the late induction of the chloramphenicol acetyltransferase gene from ori456 OCAT, while expression from inv456 OCAT increased. Site-specific mutations in two previously identified important regulatory elements of the 1.2-kb RNA promoter, the AP1-binding site and the CATA site, indicated that these sites continue to contribute to promoter activity at late times but that the replication-dependent late induction acts independently of these sites. Possible mechanisms underlying the late induction are discussed.
Wade
E J
EJ
Department of Biology, University of California, San Diego, La Jolla 92093-0116.
Spector
D H
DH
D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D004261:DNA Replication / Q000187:drug effects; D005347:Fibroblasts; D015774:Ganciclovir / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral; D006801:Humans; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012867:Skin; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; D012333:RNA, Messenger; D012367:RNA, Viral; D015500:Chloramphenicol O-Acetyltransferase; D015774:Ganciclovir