A genetic and biochemical characterization of the genes and encoded proteins that comprise the capsids of herpes simplex virus type 1 (HSV-1) is proposed. The goals of the experiments are to identify the steps in the capsid assembly pathway, to determine the role of each protein in the assembly process, to identify the molecular interactions for each capsid protein, and to locate the interactive amino acid sequences for two of the capsid proteins: VP5 and VP23. The kilodalton (kD) are VP5(150kD), VP19C (52kD), VP21 (44 kD), VP22a(40kD), VP23 (33 kD), VP24 (25kD), and VP26 (12 kD) and VP26 (12kD).
Specific Aim 1 : An immediate goal of the proposed experiments was to identify the open reading frames (ORF) that specify VP21 and VP24 and this has been accomplished. Antibodies specific for each of the capsid proteins will be isolated.
Specific Aim 2 : Transformed cells lines that express each capsid protein and null mutations in each capsid gene will be isolate.
Specific Aim 3 : Infection of Vero cells with each of the mutants should allow the accumulation of capsid precursors which will be analyzed by electron microscopy, SDS-PAGE, and hydrodynamic methods.
Specific Aim 4 : Sedimentation analysis, chemical cross-linking as well as in vitro and n vivo protein-protein binding assays will be used to identify intra-and inter- molecular interactions for each capsid protein.
Specific Aim 5 : A set of polypeptide chain termination mutants will be isolated in the genes that encode VP5 and VP23. A complementation inhibition assay will be used to fine map the interaction sites for the two proteins, and the results will be confirmed using one of the protein- protein binding assays. Mutants with different phenotypes will be incorporated into viruses to determine the effect of the missing sequences of VP 5 or VP 23 on capsid formation. The major practical application that may result from these studies is the development of antiviral compounds that mimic capsid protein interaction sites and thereby prevent formation of biologically active capsids. The finding that one of the capsid proteins, VP24, has protease activity adds another dimension to the development of antiviral.
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