Role of the UL4 protein in equine herpesvirus 1 gene programming and generation of defective interfering particles

Equine herpesvirus 1 (EHV-1) is a major pathogen of equines worldwide and provides a model for the study of alphaherpesvirus gene regulation, pathogenesis, and persistent infection mediated by defective interfering particles. The research in this dissertation focuses on the characterization of the UL4 and UL3 genes that are conserved within the genome of defective interfering particles with the goal of defining their role(s) in both lytic and persistent infection. The UL4 and UL3 genes are trans-activated by the sole immediate early (IE) protein, and metabolic inhibitor studies confirmed that both are early genes. The UL4 protein localized within the nucleus and cytoplasm of infected cells but was not detected in purified virions; whereas, the UL3 protein is a component of the tegument of purified virions. Mutant viruses that lack production of the UL4 or UL3 protein were capable of replication in a variety of cell types, demonstrating that neither protein was required for lytic replication, though smaller plaques were produced in the absence of the UL4 protein. Additionally, mice infected with the UL4- or UL3-null virus displayed symptoms of severe respiratory disease and succumbed to infection similar to mice infected with wild-type EHV-1. The UL4 protein was capable of inhibiting reporter gene expression under the control of EHV-1 promoters from all three viral gene classes as well as heterologous viral and cellular promoters. Transcripts from all three viral gene classes were increased in cells infected with the UL4- null EHV-1 as compared to infection with wild-type virus. The transcription inhibition did not depend on DNA binding, as the UL4 protein was incapable of binding viral promoter DNA. However, the inhibition of transcription appeared to be mediated through direct interactions between the UL4 protein and TATA box-binding protein and/or RNA polymerase II. Serial high multiplicity passage of an EHV-1 that lacked the complete UL4 open-reading frame demonstrated that the UL4 DNA sequence, but not a functional UL4 protein, was necessary for the generation of defective interfering particles. Collectively, these findings broaden our understanding of the role of the UL4 and UL3 proteins in both lytic and persistent EHV-1 infection.