Deborah H. Spector Adjunct Professor of Biology, UCSD e-mail: dspector@ucsd.edu Lab Homepage: Spector Lab

     A major part of our research has focused on the regulation of HCMV early gene expression. Of particular interest is our finding that the key viral transactivator, an HCMV immediate early (IE) protein designated IE2 86, is multifunctional. It binds to DNA in a site-specific (but sequence-tolerant) manner, counters histone-mediated repression of transcription, and can interact with multiple cellular proteins regulating RNA pol I and pol II transcription. IE2 86 also can form a complex with Rb and counter some of its growth-suppression functions. We also have data which suggest that DNA sequences containing the IE2 86 binding site not only modulate the level of early RNAs, but also are essential for activating late gene transcription in a distance-dependent manner. In progress are studies utilizing both in vivo genetic analyses and in vitro biochemical assays to dissect the molecular regulation of these interactions. 

     A second major area of study has evolved from our finding that the HCMV infection markedly affects key components of the cell cycle, resulting in cell cycle arrest. Early after infection, HCMV induces elevated levels of cyclins E and B, p53, and hyperphosphorylated Rb. However, the synthesis of cyclin A is inhibited, and only at very late times in the infection is there any increase in the levels of protein and kinase activity. This inhibition of cyclin A gene expression correlates with a block in host
cell DNA synthesis, and the data suggest that active cellular DNA synthesis is incompatible with the early stages of viral replication. We also have evidence that the enhanced expression of cyclin E and the inhibition of cyclin A are due to effects at the level of transcription. In contrast, the increased levels of cyclin B appear to be due to lack of cell-cycle mediated degradation. Preliminary data also indicate that during the infection there is a redistribution of the proteasomes and their overall level increases. During this past year, we have also made the interesting finding that p53 and the DNA replication protein RPA, but not Rb, become sequestered in viral replication centers in the nucleus. In addition, the nucleotide excision repair proteins XPB and XPD are also sequestered. Currently in progress are studies to address the question of whether HCMV may sequester these proteins for its own repair processes at the expense of the host. Taken together, the results of the above studies suggest that the HCMV infection has altered the transcription, subcellular localization, and stability of key cell cycle and DNA repair regulatory proteins. Moreover, the interaction between HCMV and the host cell cycle is not unidirectional, as it also appears that the phase of the cell cycle at the time of infection markedly influences the initiation of viral gene expression. 

     A third area of research relates to the in vivo pathogenesis of cytomegalovirus, using murine cytomegalovirus (MCMV) as the model. Previously, we defined the molecular biology of this virus and its pattern of replication during acute and latent infection. Our most recent studies have been directed at applying this knowledge to the development of a vaccine that will protect against
not only morbidity and mortality, but also acute and latent infection. As one approach, we are using "naked DNA" where the MCMV genes are under the control of a strong eukaryotic promoter. We have already demonstrated that intradermal inoculation of DNA encoding a MCMV IE protein confers protection, albeit not complete, and have preliminary data showing protection by a second protein. Studies are in progress to identify other protective MCMV proteins and to optimize the
vaccination protocol. 

      Rodems, S.M., Clark, C.L. and Spector, D.H. (1998). Separate DNA elements containing ATF/CREB and IE2-86 binding sites differentially regulate the human cytomegalovirus UL112-113 promoter at early and late times in the infection. J. Virol. 72:2697-2702. 

      Salvant, B.S., Fortunato, E.A. and Spector, D.H. (1998). Cell cycle dysregulation by human cytomegalovirus: influence of the cell cycle at the time of infection and effects on cyclin transcription. J. Virol. 72:3729-3741. 

      Fortunato, E.A., Sommer, M.H., Yoder, K. and Spector, D.H. (1997). Identification of domains within the human cytomegalovirus major immediate-early 86-kilodalton protein and the retinoblastoma protein required for physical and functional interaction with each other. J. Virol. 71:8176-8185. 

      Gonzalez Armas, J. C., Morello, C.S., Cranmer, L.D. and Spector, D.H. (1996). DNA immunization confers protection against murine cytomegalovirus infection. J. Virol. 70:7921-7928.