Human cytomegalovirus DNA replicates after early circularization by concatemer formation, and inversion occurs within the concatemer.
To determine the replicative mechanism for human cytomegalovirus (HCMV) DNA, field inversion gel electrophoresis was used to separate HCMV replicative DNAs during lytic infection. Unit-length circular HCMV genomes lacking terminal restriction fragments were detected starting 4 h after infection even when cells were treated with aphidicolin, phosphonoacetic acid, or cycloheximide. Viral DNA synthesis began 24 h after infection and produced large amounts of high-molecular-weight replicative DNA that was a precursor of progeny genomes. Replicative DNA contained rare terminal restriction fragments, and long-arm termini were much less frequent than short-arm termini. Replicative DNA was not composed of unit-length circles because low-dose gamma irradiation of replicative DNA generated numerous random high-molecular-weight fragments rather than unit-length molecules. PacI digestion of replicative DNA from a recombinant HCMV with two closely spaced PacI sites revealed that replicative DNA is concatemeric and genome segment inversion occurs after concatemer synthesis. These results show that after circularization of the parental genome, DNA synthesis produces concatemers and genomic inversion occurs within concatemeric DNA. The results further suggest that concatemers acquire genomic termini during the cleavage/packaging process which preferentially inserts short-arm termini into empty capsids, causing a predominance of short-arm termini on the concatemer.
McVoy
M A
MA
Department of Pediatrics, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298.
Adler
S P
SP
D016590:Aphidicolin / Q000494:pharmacology; D002478:Cells, Cultured; D007446:Chromosome Inversion; D003513:Cycloheximide / Q000494:pharmacology; D003587:Cytomegalovirus / Q000235:genetics* / Q000254:growth & development; D004261:DNA Replication / Q000187:drug effects; D004270:DNA, Circular / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D004274:DNA, Recombinant / Q000378:metabolism; D004279:DNA, Viral / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005347:Fibroblasts / Q000382:microbiology; D005720:Gamma Rays; D016679:Genome, Viral; D006801:Humans; D007553:Isotope Labeling; D008836:Micrococcal Nuclease / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D010746:Phosphonoacetic Acid / Q000494:pharmacology
D004270:DNA, Circular; D004274:DNA, Recombinant; D004279:DNA, Viral; D016590:Aphidicolin; D003513:Cycloheximide; D015252:Deoxyribonucleases, Type II Site-Specific; D008836:Micrococcal Nuclease; C068375:endodeoxyribonuclease PacI; D010746:Phosphonoacetic Acid