The Capsid of herpes simplex virus type 1 (HSV-1) is an icosahedral (T=16) protein shell approximately 15 nm thick and 125nm in diameter. In the intact virus, the capsid contains the viral dsDNA and it is surrounded by a lipid membrane. The goal of this project is to assemble the HSV-1 capsid in vitro beginning with purified capsid proteins. Capsids will be isolated from the nuclei of HSV- 1- infected cells and dissociated into their six component protein with dissociating/denaturing agents such as guanidine-HC1 or urea. Individual capsid proteins will then be purified and added together under conditions designed to promote their re-assembly into icosahedral capsids. Reaction mixtures will be examined for the presence of re-assembled capsids by electron microscopy, sucrose density gradient ultracentrifugation and SDS-polyacrylamide gel electrophoresis. Also, studies involving the use of specific monoclonal antibodies will be carried out to define the locations of capsid proteins VP19, VP23, and VP26. Specific goals of the project are to: (1) define the morphology, total mass and protein stoichiometry of torus-shaped structures self-assembled in vitro from purified capsid protein VP22a; (2) identify conditions in which native VP26 can re-attach to HSV-1 capsids that lack it and define the location of VP26 in reconstituted capsids; (3) define conditions in which purified VP, the major capsid protein, can assembly in vitro into T=16 icosahedra. Assembly may require the presence of other capsid proteins such as the scaffolding protein, VP22a: and (4) prepare monoclonal antibodies specific for capsid proteins VP19, VP23, and VP26, and use them (in conjunction with electron microscopic methods) to localize their cognate proteins in the HSV-1 capsid.
