Generation and characterization of an EICP0 null mutant of equine herpesvirus 1

The EICP0 gene (gene 63) of equine herpesvirus 1 (EHV-1) encodes an early regulatory protein that is a promiscuous trans-activator of all classes of viral genes. Bacterial artificial chromosome (BAC) technology and RecE/T cloning were employed to delete the EICP0 gene from EHV-1 strain KyA. Polymerase chain reaction, Southern blot analysis, and DNA sequencing confirmed the deletion of the EICP0 gene and its replacement with a kanamycin resistance gene in mutant KyA. Transfection of rabbit kidney cells with the EICP0 mutant genome produced infectious virus, indicating that the EICP0 gene is not essential for KyA replication in cell culture. Experiments to assess the effect of the EICP0 deletion on EHV-1 gene programming revealed that mRNA expression of the immediate-early gene and representative early and late genes as well as the synthesis of these viral proteins were reduced as compared to the kinetics of viral mRNA and protein synthesis observed for the wild type virus. However, the transition from early to late viral gene expression was not prevented or delayed, suggesting that the absence of the EICP0 gene did not disrupt the temporal aspects of EHV-1 gene regulation. The extracellular virus titer and plaque areas of the EICP0 mutant virus KyADeltaEICP0, in which the gp2-encoding gene 71 gene that is absent in the KyA BAC was restored, were reduced by 10-fold and 19%, respectively, when compared to parental KyA virus; while the titer and plaque areas of mutant KyADeltaEICP0Deltagp2 that lacks both the EICP0 gene and gene 71 were reduced more than 50-fold and 67%, respectively. The above results show that the EICP0 gene is dispensable for EHV-1 replication in cell culture, and that the switch from early to late viral gene expression for the representative genes examined does not require the EICP0 protein, but that the EICP0 protein may be structurally required for virus egress and cell-to-cell spread.