Evidence that the UL84 gene product of human cytomegalovirus is essential for promoting oriLyt-dependent DNA replication and formation of replication compartments in cotransfection assays

Abstract

The protein products of 11 viral genomic loci cooperate in a transient cotransfection assay to mediate lytic-phase DNA replication of oriLyt, the human cytomegalovirus (HCMV) origin of replication. Six of these genes have homology with the well-characterized herpes simplex virus replication genes and encode core replication machinery proteins that are typically essential for DNA synthesis. The remaining five HCMV gene loci, initially referred to as auxiliary components, include several known immediate-early (IE) transcriptional regulatory proteins as well as genes encoding functionally uncharacterized polypeptides. Some or all of the auxiliary components may be necessary in trans to replicate the HCMV oriLyt only because they are required for efficient expression or transactivation of the native early promoters and 3 processing elements included in the genomic clones. Therefore, we reassessed the requirements for the auxiliary components by adding constitutive heterologous promoters and control signals to the coding regions and carrying out transient DpnI replication assays in cotransfected Vero cells. The results revealed that in the presence of the UL69 posttranscriptional activator and the remaining auxiliary polypeptides, UL84 was the only auxiliary component that could not be omitted to obtain oriLyt-dependent DNA replication. Nevertheless, in human diploid fibroblasts, some additional auxiliary loci as well as UL84 were critical. There was also an obligatory requirement for UL84, in cooperation with two other auxiliary factors, UL112-113 and IE2, and the core machinery, to constitute the minimal HCMV proteins necessary to direct oriLyt-dependent DNA amplification. However, the Epstein-Barr virus core replication genes could substitute for the HCMV core genes, and in these circumstances, UL84 alone directed amplification of HCMV oriLyt. Moreover, there was also an absolute requirement for UL84 along with the core and other auxiliary factors for the formation of intranuclear replication compartments as assayed by immunofluorescence in transient DNA cotransfection assays. These compartments were typical of those associated with active viral DNA replication in HCMV-infected cells, they incorporated pulse-labeled bromodeoxyuridine, and their formation was both phosphonoacetic acid sensitive and oriLyt dependent. These results demonstrate that UL84 is obligatory for both intranuclear replication compartment formation and origin-dependent DNA amplification and suggest that it is a key viral component in promoting the initiation of HCMV oriLyt-directed DNA replication.

Recombination in viruses: Mechanisms, methods of study, and evolutionary consequences

“…Moreover, all Herpesviruses have a common replicative complex characterized by highly conserved groups of replication and recombination proteins (Rennekamp and Lieberman, 2010). In fact, many of these proteins can be interchanged between different Herpesviruses; for example, Epstein-Barr virus proteins have been shown to replicate human cytomegalovirus (Sarisky and Hayward, 1996). In DNA viruses, recombination also plays an important role in the repair of DNA damage, especially in the latency stage (Wilkinson and Weller, 2004) but, paradoxically to previous observations, the analysis of complete genomes of many Herpesviruses has shown that the repair-initiating DNA sequences differ between aand c-Herpesviruses, thus suggesting possible differences in the recombination mechanisms between these two subfamilies (Brown, 2014).…”

Identification of Persistent RNA-DNA Hybrid Structures within the Origin of Replication of Human Cytomegalovirus

“…Origin-dependent replication of HCMV in primary human cells requires the gene products of the core replication open reading frames in addition to UL36-38, UL112-113, IE1/IE2 and UL84 (22,27). In Vero cells, however, UL84 appears to be the only non-core replication protein required for oriLyt-dependent replication (27).…”

“…Initially, UL84 was shown to be 1 of 11 loci required for oriLyt-dependent DNA replication (22). More recently, this observation was underscored by Sarisky and Hayward, who demonstrated that UL84 plays a critical role in transient replication (27). The exact function of the UL84 protein in DNA replication remains unknown.…”

A replication function associated with the activation domain of the Epstein-Barr virus Zta transactivator

“…After PCR amplification, the DNA product was cleaved with the appropriate restriction endonucleases and subcloned into pRTS2 as follows: pRTS11, -12, -13, -14, -15, -16, -25, and -28 as XbaI-HindIII fragments and pRTS21 as a BamHI-BglII fragment. These plasmid constructions have been described previously (58), as has the origin-containing plasmid, pSL77 (19).…”