HERPESVIRUS PROTEINASE--TARGET FOR ANTIVIRAL THERAPY

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A cytomegalovirus gene encoding a maturational proteinase has been identified, cloned, and sequenced. The gene is 1,770 bp in length and gives rise to a protein that has an M of approximately 85 kDa, as estimated by polyacylamide gel electrophoresis. The enzyme active site appears to be located in the amino one-half of the protein -- probably within two domains that are highly conserved in the homologous proteins of other herpesviruses. The substrate of this proteinase is a protein referred to as the assembly protein precursor. There is evidence that cleavage of the precursor to the mature assembly protein is essential for herpesvirus maturation. Therefore, interference with this cleavage event would be expected to have a potent antiviral effect. The specific site at which the viral proteinase cleaves its substrate has been determined, and a transfection assay utilizing the cloned genes for the proteinase and its substrate has been developed to monitor the cleavage event. Studies proposed in this application are intended to further characterize this herpesvirus maturational proteinase, and to evaluate its potential as a new target for antiviral drug development. Five specific aims are listed which seek to (i) verify that the enzyme is freed from a much larger precursor by cleavage at a putative consensus enzyme "release" site; (ii) identify the functionally important amino acids in both the enzyme "release" and substrate "maturational" cleavage sites, and determine what features distinguish the two; (iii) delineate the active site domain of the proteinase and identify functionally important amino acids within that region; (iv) develop a cell-free system to permit quicker, more versatile, and quantitative assays of the proteinase; and (v) produce large amounts of the active viral proteinase for biochemical and crystallographic studies. Results of this work are anticipated to provide valuable leads for developing perhaps broad spectrum antiherpesvirus drugs targeted to the maturational proteinase. It is also likely that useful new information will be gained in the general area of viral proteinase mechanisms, and in the specific area of herpesvirus replication.