Herpes-group viruses encode a serine maturational proteinase that is essential for the production of infectious progeny. The cytomegalovirus (CMV) homologue of this protein is called assemblin and is encoded by the UL80a open reading frame in human CMV. Assemblin is synthesized as a precursor (~74kDa) that undergoes four sequential autoproteolytic cleavages. The four cleavage sites have five amino acid, core consensus sequences that are well conserved among their counterparts in other herpes virus. The principal substrate of assemblin is an abundant capsid assembly protein that also contains the M-cleavage site at its carboxyl end, as a consequence of its interesting in-frame, nested genetic relationship with the proteinase. Because these cleavages are essential for virus production, inhibition of the proteinase would be expected to have a potent antiviral effect. New drugs with antiviral activity against herpes-group viruses are needed, and the studies proposed in this application are intended to further characterize the physical, enzymatic, and biological properties of the herpesvirus proteinase, in particular the CMV enzyme, and help exploit it as an effective molecular target for drug development.
The specific aims of the work proposed in this competitive renewal application are: (i) prepare proteinase and assembly protein precursors for use in enzyme assays and crystallography; ii) develop an in vitro proteinase assay that uses """"""""native"""""""" substrate' (iii) determine what cleavage-site features influence cleavage kinetics; (iv) identify the interactive domains of two chain assemblin; (v) collaborate to study and compare gamma-2 HHV8 assemblin with its alpha (e.g., herpes simplex virus) and beta (e.g., CMV) herpesvirus homologues; (vi) test potential antivirals in cell culture; and (vii) determine the ability of virus to """"""""escape"""""""" their effect. Results of this work are anticipated to provide useful new information about this apparently novel member of the serine proteinase family, and contribute to the development of inhibitors that will block its function. It is also likely that useful new information will be generated in the areas of viral proteinase mechanisms and herpesvirus replication.
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