Herpes simplex virus (HSV) remains a major pathogen world-wide causing human diseases including cold sores, eye and genital infections, neonatal infections, and encephalitis. HSV establishes a life-long latent infection which reactivates periodically to produce recurrent disease. The virus envelope contains ten glycoproteins that are important for infection and pathogenesis. This grant focuses on the structure-function relationship of two of the glycoproteins, gD and gB, both of which are crucial for viral entry and are in Phase III trials as human vaccines. Few details are known about the entry process or about structural changes that occur to the glycoproteins during entry. Our goal is to better define the structures of gD and gB, and to understand how structure is related to function.
In Aim 1, three approaches will be used to probe gD structure at increasing levels of resolution; (i) antigenic and biophysical studies; (ii) visualization by EM methods including scanning transmission electron microscopy and 2-D electron crystallography; and (iii) crystallization of gD and of gD complexed with MAb DL11 leading to X-ray diffraction analysis. The DL11 epitope overlaps a functional site as well as a virus neutralizing site on gD. All of the studies in Aim 1 will employ biologically active and antigenically correct, truncated forms of gD-1 and gD-2 obtained from baculovirus recombinants.
In Aim 2 the recombinant proteins and non-functional but antigenically active mutants will be used in in vitro assays to dissect the effects of the mutations on entry. We will look at the effect of mutations on the ability of gD to bind to cells, block HSV infection, interact with the mannose-6-phosphate receptor, and function in fusion. The goal is to understand what regions of gD are needed for receptor binding as well as to define the regions of gD involved in any additional role played by the protein in virus infection.
Aim 3 focuses on a structure-function analysis of gB. Dimeric gB found in virus-infected cells and in virions is closely associated with fusion leading to virus entry and cell to cell spread. Our hypothesis it that a """"""""triggering"""""""" event causes conformational changes in dimeric gB resulting in fusion between the virion envelope and the cellular membrane. We will examine: (i) gB purified from HSV-infected cells, or truncated and purified from a baculovirus expression system; (ii) gB as it exits in virions; (iii) gB as it exists in the cell surface; and (iv) the role of gB in fusion using a new assay. These studies will involve the use of a panel of MAbs, some of which recognize only the dimeric form of gB. Thus the specific aims are: (l) to study the immunological and biophysical properties of HSV gD; (2) to relate structural properties of gD to its function in virus infection; and (3) to relate structural properties of HSV gB to its function in virus infection.
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