The envelope glycoproteins of herpesviruses play critical roles in the initial virus adsorption to a target cell, and in the subsequent envelopment, egress, and release of progeny virons. To perform these functions, the viral glycoproteins must use the existing cellular protein export machinery to achieve a proper membrane or envelope localization. Pseudorabies virus (PRV) is a major pathogen of swine, and has been the subject of several vaccines. In each case, the glycoproteins have been the focus for attenuation. The proposal concerns the identification of PRV glycoprotein export information as an approach to better understand the structure/function relationship of herpesvirus glycoproteins. The signal sequence for glycoprotein gIII, a major yet nonessential component of the PRV envelope, is absolutely required for gIII's export and proper function. Each structural domain of the gIII signal sequence will be the target of site-directed mutagenesis experiments to ascertain the role of each domain in the correct initiation of export. The hydrophilic character of the amino- terminus will be reduced, and the hydrophobic and alpha-helical properties of the central core will be disrupted. Additionally, the consensus signal peptidase processing sites will be altered. The effect of each change will be determined through the construction of recombinant viruses harboring the signal sequence mutations. Those mutants sufficient blocked in export will be used in a reversion analysis to further analyze the adverse effects of the signal sequence alterations. Revertants will be obtained through their superior ability to adsorb to tissue culture monolayers. While variable in tissue culture, gIII-deficient viruses exhibit growth defects, including inferior abilities to attach to, and be released from, host cells. This phenotype will be exploited as noted for export studies, and will also be the focus of a mutational analysis of the mature body of gIII. Deletions and small insertions will be introduced throughout a cloned copy of the gIII gene, and selected classes will be recombined into the virus genome. Recombinant virus will be assayed for their ability to adsorb to host cells, and their gIII species will be tested for export competence. In this manner, gIII functional domains should be identified. Lastly, a physical characterization of gIII, including a determination of whether or not gIII assumes a multimeric state in the infected cell will lend further insight into the relationship of gIII structure and its functions.
