Deborah H. Spector
e-mail: dspector@ucsd.edu |
 |
Human cytomegalovirus (HCMV), a herpesvirus, is the leading viral cause of birth defects and poses a serious health threat to immunocompromised individuals. The long term goal of our work is to determine at the molecular level how the interplay of viral and host functions relates to the in vivo pathogenesis, and to use this information to develop effective strategies for treatment and prevention of disease.
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.
Fortunato, E.A. and Spector, D.H. (1998). p53 and RPA are sequestered in viral replication centers in the nuclei of cells infected with human cytomegalovirus. J. Virol. 72:2033-2039.
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.
Deborah Spector received her Ph.D. from the Massachusetts Institute of Technology. She was a Helen Hay Whitney Foundation Postdoctoral Fellow at UCSF. Professor Spector received Kaiser Permanente Teaching Awards in 1983 and 1985.
