Replication of Epstein-Barr virus oriLyt: lack of a dedicated virally encoded origin-binding protein and dependence on Zta in cotransfection assays

Abstract

Using a transient replication assay in which cosmid DNAs were cotransfected into Vero cells, we had previously demonstrated that oriLyt replication required six Epstein-Barr virus (EBV)-encoded replication genes. No oriLyt origin-binding protein was identified in this study, but oriLyt replication in the cotransfection assay was also dependent on the three lytic cycle transactivators Zta, Rta, and Mta and an activity encoded by the EBV SalI F fragment. We have now used expression plasmids for the six known replication proteins to further examine the question of the requirement for an oriLyt origin-binding protein. The activity in SalI-F was shown to be encoded by BKRF3. The predicted product of this open reading frame is an enzyme, uracyl DNA glycosylase, not an origin-binding protein, and is dispensable for replication in assays using expression plasmids. BBLF2, which is positionally related to the gene for the herpes simplex virus (HSV) UL9 originbinding protein, was confirmed to be expressed as a spliced transcript with BBLF3 and not as an independent product. Examination of the requirement for the EBV transactivators revealed that Rta, while contributing to replication efficiency, was dispensable. Mta could be substituted by HSV IE63, and in complementation experiments with HSV replication genes, Mta was no longer required for replication of EBV oriLyt, suggesting that the contribution of Mta to replication may be indirect. Zta continued to be required for detectable oriLyt replication both with the EBV replication proteins and in the complementation assays with HSV replication proteins. We conclude that EBV does not encode an equivalent of HSV UL9 and that Zta is the sole virally encoded protein serving an essential origin-binding function.

Cellular transcription factors recruit viral replication proteins to activate the Epstein–Barr virus origin of lytic DNA replication, oriLyt

“…Six EBV-encoded gene products are required to execute lytic DNA replication at the level of DNA synthesis: the DNA polymerase (BALF5); the polymerase processivity factor (BMRF1); the single-stranded DNA-binding protein (BALF2); the primase (BSLF1); the primase-associated protein (BBLF2/3); and the helicase (BBLF4) (Fixman et al, 1992(Fixman et al, , 1995. In transfected cells, these six viral gene products together with BZLF1 are sufficient for the replication of oriLyt-containing plasmids.…”

Four of eleven loci required for transient complementation of human cytomegalovirus DNA replication cooperate to activate expression of replication genes

“…The finding that five additional loci were needed to complement HCMV DNA replication was surprising because homologous proteins were not required to complement HSV-1 or Epstein-Barr virus DNA synthesis (16,17,43). Moreover, none of these five showed evidence of similarity to the seventh essential HSV-1 replication component, the origin-binding protein UL9 (8,13,14).…”

Herpes Virus Replication

“…The initiation process in other herpesviruses is less well understood and possibly more complicated. In EBV for example, it is known that Rta and Zta proteins are required for lytic cycle induction (70)(71)(72). Zta has also been shown to possess oriLyt-binding activity and to recruit other replication factors (helicase-primase and SSB) presumably to initiate assembly of a replisome (71).…”

“…KSHV transactivator ORF50 (EBV Rta homologue (72)) induces the viral lytic cycle (99). Additional factors such as ORF-K8 (EBV Zta homologue (70,71)) may also be required for oriLyt-dependent DNA replication (49). 5 It is thought that the mature protein is formed from a spliced transcript involving these ORFs.…”