Herpes simples viruses cause a variety of human diseases, including cold sores, eye and genital infections, neonatal infections and encephalitis. The virion envelope contains a number of glycoproteins which play important roles in HSV infection and pathogenesis. This grant is focused on glycoprotein D, which is essential for virus entry and is important for HSV neuroinvasiveness. Structural and immunological properties of gD make it an important candidate for a subunit vaccine against infections by HSV-1 (oral) and HSV-2 (genital). The objective of this study is to define the functions of gD and to relate them to its structure. The major approach in this renewal application is to construct mutations in the gD gene by site-directed mutagenesis and to test the ability of the altered gD protein to function biologically by use of a complementation assay. The structure of the mutated proteins expressed in transfected mammalian cells will be examined with a panel of monoclonal antibodies. Other properties of the expressed proteins, including aggregation, processing and transport to the cell surface will also be investigated. Changes which alter the ability of gD to function, but otherwise have little or no effect on these other properties, will identify important functional regions. Certain mutations in the gD gene will be recombined into the virus genome and the effect of these mutations on the phenotype of the recombinant viruses will be assayed in cell culture and in an animal model of neuroinvasion. There is good evidence that gD interacts with a specific cell receptor as an important step leading to virus-cell fusion, and two broad approaches will be used to examine this issue further. First, experiments will be carried out to determine if the initial interaction between HSV and the cell involves alterations in gD conformation, or in the conformation of other HSV glycoproteins. Experiments will examine the state of the glycoproteins immediately after infection with radiolabeled purified virus. In the second approach, experiments are designed to identify and characterize the cell surface molecule (receptor) with which gD interacts. The major experimental approach will be the use of a gD affinity column to isolate the cell receptor.
The specific aims are: 1) to analyze the effect of specific mutations in the gD gene on the conformation, biological properties and function of the protein; 2) to determine if the conformation of gD or other viral glycoproteins changes as a result of virus entry; 3) to determine if gD mutations affecting cell entry also influence neuroinvasiveness; and 4) to identify cell surface molecules (receptors) which interact with HSV gD.
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