Herpesviruses are important pathogens, and information on their replication is useful in the diagnosis and treatment of these infections. Our goals are to employ our models of equine herpesvirus 1 (EHV) cytocidal and persistent infection to understand viral replication in terms of structure/function relationships of the viral regulatory proteins and to ascertain if their interactions with cellular proteins influence the outcome of infection. In years #44 to #48, we seek to: i. define the mechanism of antagonism between the immediate early protein (IEP) and the early EICP0 protein that may control the switch to late gene expression and antagonize the IEP by competition for transcription factors TBP, TFIIB; ii. ascertain which transcription factors interact with the IEP and early regulatory proteins EICP0P, EICP22P and EICP27P; iii. characterize our +17 IE mutant EHV and use our Bacterial Artificial Chromosome plasmids to generate additional EHV that express mutated regulatory proteins so that their functions may be identified and mapped; iv. determine how EICP22 and EICP27 proteins synergize the IEP to activate viral gene expression by assessing if physical interactions occur between these EHV protein(s) and cellular transcription factors, or if post-transcriptional mechanisms operate, e.g. enhancing transport of EHV transcripts. We shall address the mechanism of EHV persistent infection mediated by defective interfering particles (DIP) that express only the UL1, UL2 and a unique hybrid protein (HYB) comprised of portions of the EICP22 and EICP27 proteins and reduce EHV virulence in the animal. DIP mutants that vary in these ORFs will be assayed for the capacity to interfere with EHV replication, establish persistent infection, and alter EHV gene regulation. Information on the EICP22P and EICP27P domains in the HYB will give insight into how the HYB alters EHV gene programming, such as by functioning as a dominant negative form of the EICP22 and EICP27 proteins. Microarray technology will define the changes in cell gene expression in EHV infection versus DIP infection and persistent infection. In all the above experiments, emphasis will be given to assess the biological properties of the mutant viruses in infected cells; mutants of interest will also be assessed in the mouse model. Lastly, EHV with mutations in glycoproteins gI and gE will be assessed in our mouse model to learn more about EHV pathogenesis and to test our hypothesis that EHV lethality is due to immunopathology mediated by proinflammatory cytokine and chemokine production associated with the expression of the gI and/or gE proteins.
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