During the initial stages of lytic infectious cycle of herpes simplex virus (HSV-1, a widespread human pathogen), transcription of the viral immediate-early (IE) genes is specifically and potently activated by a virion protein termed VP16. The broad objective of this proposal is to uncover details of he mechanism of this trans-activation. The initial strategy for accomplishing this objective is to discern the cis-acting DNA sequence elements which respond to activation, and to characterize functional domains of the trans-activation proteins VP16. Previous work on the IE gene encoding the ICP4 protein revealed the presence of two distinct cis-response elements which mediate the action of VP16. However, VP16 does not bind directly to these elements. Instead, the action of VP16 is apparently mediated by two host cell proteins which do bind specifically to he two cis elements. Sequences resembling the ICP4 cis-response elements can be found in the regulatory regions of the other IE genes. One hypothesis to be explicitly tested in this proposal is that those related sequences also mediate VP16 trans-activation. Putative response elements will be altered using site-directed mutagenesis. Mutant plasmids will be tested in transient expression assays for th ability to respond to VP16. A corollary hypothesis is that the host proteins which bind to the ICP4 cis-response elements will also bind to the response elemens of other IE genes. This hypothesis will be tested by DNAse I footprinting and gel mobility shift assays using partially purified host DNA binding proteins. Previous work ahs also begun to identify functional domains of the VP16 activating protein. An acidic carboxyl termianl domain is critical for activaton, and is likely to be the physical link to the transcriptional machinery.
A specific aim of this proposal is to identify individual amino acid residues which play major roles in this function, using site-directed mutagenesis of the VP16 gene. Boundaries of VP16 doamins which interact with the tow host cell DNA-binding factors have also been identified. This proposal seeks to use deletion and amino acid substitution mutagenesis to more clearly define the domains for these protein:protein interactions. The initial identification and purification of the host factors is beyond the scope of theis proposal, and is being actively pursued in other laboratories. Once identified, however, biochemical and immunological techniques will be employed to directly investigate the interactions of wildtype an mutant VP16 proteins with the associated host factors.
| Shen, F; Triezenberg, S J; Hensley, P et al. (1996) Transcriptional activation domain of the herpesvirus protein VP16 becomes conformationally constrained upon interaction with basal transcription factors. J Biol Chem 271:4827-37 |
| Shen, F; Triezenberg, S J; Hensley, P et al. (1996) Critical amino acids in the transcriptional activation domain of the herpesvirus protein VP16 are solvent-exposed in highly mobile protein segments. An intrinsic fluorescence study. J Biol Chem 271:4819-26 |
