Analysis of Human Cytomegalovirus ori Lyt Sequence Requirements in the Context of the Viral Genome

Abstract

ABSTRACT During the lytic phase of infection, replication of herpesvirus genomes initiates at the lytic origin of replication, ori Lyt. Many herpesviruses harbor more than one lytic origin, but so far, only one ori Lyt 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 ori Lyt was described as a core region of 1.5 kbp (minimal ori Lyt) flanked by auxiliary sequences required for maximal replication activity (complete ori Lyt). It remained unclear whether minimal ori Lyt alone can drive the replication of HCMV in the absence of its accessory regions. To investigate the sequence requirements of ori Lyt in the context of the viral genome, mutant genomes were constructed lacking either minimal or complete ori Lyt. These genomes were not infectious, suggesting that HCMV contains only one lytic origin of replication. Either minimal or complete ori Lyt was then ectopically reinserted into the ori Lyt-depleted genomes. Only the mutant genomes carrying complete ori Lyt 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 ori Lyt plus the left flanking region gave rise to minifoci, but genomes harboring minimal ori Lyt 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 ori Lyt.