Mouse adenovirus type 1 (MAV-1) provides a model system for studying the molecular basis of adenoviral pathogenesis, which cannot be pursued using the more extensively characterized human adenoviruses. MAV-1 causes acute and persistent infections, and the effects of viral mutations on the infectious process can be studied in the natural animal host. During the previous funding period we established the basic organization of the MAV-1 genome, and determined DNA sequences and transcription maps of two key early MAV-1 genes, early region 1A (E1A) and early region 3 (E3). We also developed an approach for in vitro site-specific mutagenesis of the MAV-1 genome. We now will exploit these developments to study the molecular basis of virus-host interactions for MAV-1 genes E1A and E3. The corresponding human adenovirus gene products interact with host cell components and have been postulated to have important roles in adenoviral disease. The human adenovirus E1A proteins transactivate viral and cellular genes, and form complexes with cellular proteins, including a tumor suppressor, the retinoblastoma protein (pRB). These activities have possible significance for viral pathogenesis. In human adenovirus infections an E3protein reduces expression of class I antigens of the major histocampatibility complex on the cell surface; this may be a factor contributing to adenoviral persistence. We will focus our study on the virus-host interactions by introducing specific mutations into the MAV-1 E1A and E3 genes. We have evidence that MAV-1 E1A forms a complex with pRB in vitro. We will confirm that this association is found in MAV-1- infected cells and we will construct specific mutants that disrupt this complex. We also will make mutations to eliminate another function we have identified for MAV-1 E1, transactivation of the human adenovirus E3 promoter. Mutations which alter transactivation or formation of an E1- ApRB complex will be selected on the basis of in vitro studies and transferred to the MAV-1 genome for study in the cell culture and mice. Viral mutants in E3 will be constructed to determine the importance of the MAV-1 E3 gene for viral infection and to determine whether an E3- dependent alteration in class I antigen expression occurs in MAV-1 infected cells, as it does in human adenovirus 2 infections. If so, We will test the ability of E3 mutant viruses to cause persistent infections. The influences of E1A and E3 mutations on the properties of the mutant viruses will be determined both in cell culture and mice. Early mRNA and protein synthesis, DNA replication, late protein synthesis, and interactions between host cell proteins and viral early proteins will be examined in cell culture. Effects of mutations to be studied in mice include variations in lethality, the ability to establish persistent infection, tissue specificty and progression of mutant virus infections, and the host inflammatory response. These studies should provide a unique opportunity to correlate the molecular biology of an animal virus with specific determinants responsible for viral pathogenesis.
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