Analysis of human cytomegalovirus oriLyt sequence requirements in the context of the viral genome.
During the lytic phase of infection, replication of herpesvirus genomes initiates at the lytic origin of replication, oriLyt. Many herpesviruses harbor more than one lytic origin, but so far, only one oriLyt has been identified for human cytomegalovirus (HCMV). Evidence for the existence of additional lytic origins of HCMV has remained elusive. On the basis of transient replication assays with cloned viral fragments, HCMV oriLyt was described as a core region of 1.5 kbp (minimal oriLyt) flanked by auxiliary sequences required for maximal replication activity (complete oriLyt). It remained unclear whether minimal oriLyt alone can drive the replication of HCMV in the absence of its accessory regions. To investigate the sequence requirements of oriLyt in the context of the viral genome, mutant genomes were constructed lacking either minimal or complete oriLyt. These genomes were not infectious, suggesting that HCMV contains only one lytic origin of replication. Either minimal or complete oriLyt was then ectopically reinserted into the oriLyt-depleted genomes. Only the mutant genomes carrying complete oriLyt led to infectious progeny. Remarkably, inversion of the 1.5-kbp core origin relative to its flanking regions resulted in a replication-defective genome. Mutant genomes carrying minimal oriLyt plus the left flanking region gave rise to minifoci, but genomes harboring minimal oriLyt together with the right flanking region were noninfectious. We conclude that the previously defined minimal lytic origin is not sufficient to drive replication of the HCMV genome. Rather, our results underline the importance of the accessory regions and their correct arrangement for the function of HCMV oriLyt.
Borst
Eva-Maria
EM
Virus Cell Interaction Group, ZAMED, Medical Faculty, Martin-Luther-University of Halle-Wittenberg, Halle (Saale), Germany.
Messerle
Martin
M
D024509:3' Flanking Region; D024506:5' Flanking Region; D002478:Cells, Cultured; D007446:Chromosome Inversion / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics* / Q000254:growth & development*; D004279:DNA, Viral / Q000235:genetics / Q000502:physiology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018741:Replication Origin; D017384:Sequence Deletion; D014764:Viral Proteins / Q000235:genetics* / Q000502:physiology*; D014779:Virus Replication
D004279:DNA, Viral; D014764:Viral Proteins; C112091:oriLyt protein, Human cytomegalovirus; D049452:Green Fluorescent Proteins
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