MOLECULAR GENETICS OF HSV 1 CAPSIDS

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DESCRIPTION: A genetic and biochemical study of herpes simplex virus type 1 (HSV-1) capsids is proposed. Major goals of the proposed experiments are to identify conformation changes in capsid structure that occur during B capsid maturation, to identify residues in the molecules that cause the changes, and to identify interactions between the molecules through which the changes may be mediated. Capsid shells are composed of three essential proteins; VP5, the major capsid shell component, and VP19C-VP23, a complex which interacts with and stabilizes VP5. The UL26 (VP24 and 21) and the more abundant UL26.5 (22a) gene products specify the protease activity (VP24), and the scaffold (22a and probably 21) on which the shell is assembled via interactions with VP5. Specific Aim 1. Residues of UL26 and UL26.5 gene products known to interact with VP5 will be altered that (1.1). block protease cleavage at the maturation site, or (1.2). prevent essential interactions with VP5. Compensatory genetic changes, presumably in VP5, will be selected that allow the blocks to be overcome. Specific Aim 2. Changes in shape (spherical to icosahedral) may accompany B capsid maturation. Mutant viruses possibly blocked at steps in the maturation process will be examined during infection of non-permissive cells by electron microscopy, cellular immunofluorescence using a VP5 specific antibody, and by SDS-PAGE analysis of capsid proteins following sedimentation analysis. Specific Aim 3. The size, shape and oligomeric status of the major scaffold molecule (22a) will be determined. The yeast GCN4 and HIV-1 gp160 residues that specify basic leucine zipper motifs may be used to replace the self-interactive domain. The transdominance of mutant viruses will be evaluated. Specific Aim 4. Residues of VP26 that interact with VP5 will be identified using VP26-negative capsids incubated with wild-type and mutant forms of VP26 synthesized in vitro. Specific Aim 5. Studies will be pursued to determine the structure of the VP19C-VP23 complex by x-ray crystallography. A number of insertions in VP19C coding sequences will aid the identification of residues of VP19C that interact with VP23 and with VP5. In vitro synthesized products of VP19C and VP23 will be assayed by co-immunoprecipitation and a capsid binding assay, respectively. Specific Aim 6. Experiments are proposed to package viral DNA into capsids, and to determine the direction of packaging of DNA into capsids.