Functional roles of the human cytomegalovirus IE2 86 kDa protein in HCMV-infected cells

The human cytomegalovirus (HCMV) IE2 86 kDa protein is an essential viral regulatory factor that has been shown in transient transfection and in vitro assays to transactivate early viral promoters and to interact with many viral and cellular proteins. To understand the functions provided by this protein in the HCMV-infected cell, we have constructed and characterized a family of recombinant viruses containing changes to the IE2 gene and other parts of the HCMV major immediate early (IE) region. The study of these HCMV mutants has allowed us to confirm that several of the predicted functions of IE2 86 are relevant in the virus-infected cell and has identified new functions for the protein. Introducing small deletions into the C-terminus of IE2 86 resulted in viruses that do not support early gene expression, replicate, or repress the major IE promoter. Surprisingly, these constructs also mediated up-regulation of several delayed early and late viral genes, suggesting that IE2 functions are required for the proper regulation of late viral gene expression. By constructing several viruses that do not express the IE2 40 and IE2 60 kDa proteins, which are present in infected cells at late times post infection, we continued to investigate the regulation of late gene expression by IE2. Again, we found that the IE2 40 and IE2 60 proteins are required for proper late gene expression and for repression of the major IE promoter. Interactions between the virus and the host cell are also crucial for proper HCMV replication, and a recombinant virus with a deletion in exon 3 of the major IE region demonstrated that IE2 86 is important not only for transactivation of viral early promoters, but also for dysregulation of the host cell cycle. Cells infected with this recombinant virus fail to exhibit diffuse PML staining at early times post infection and express less cyclin E protein and produce less infectious virus than do wild-type virus-infected controls. The experiments described in this dissertation demonstrate that IE2 86 is a multifunctional protein that contributes many functions, several of which were not previously identified, to HCMV replication in infected cell