For the two herpes simplex viruses (HSV-1 and HSV-2), four glycoproteins designated gB, gD and a complex of gH/gL are essential for virus entry. A fifth glycoprotein, gC, though not essential, is important for facilitating initial attachment by binding to cell surface heparin sulfate proteoglycans. gD triggers entry by interacting with one of several different cellular receptors. gB and gH/gL are necessary for fusion of the envelope with the plasma membrane. The long-term objective of the research efforts of Drs. Cohen and Eisenberg is to understand the mechanism by which these glycoproteins interact with each other and with cell molecules to mediate HSV entry. Within this proposal, they propose three specific aims: 1) to study immunological and biochemical properties of soluble gD, alone or in combination, with two of its receptors, HveA (a TNF receptor) or HveC (an adhesion molecule in the Ig superfamily); 2) to study the gD-receptor interaction when either the ligand or the receptor (or both) is membrane bound; and 3) to study events in virus entry that occur as a consequence of gD-receptor interactions. The investigators suggest that downstream interactions of the virus with the plasma membrane may involve gB and gH/gL of the virus and may additionally involve other cellular molecules. They have cloned the ectodomains of gD, gB, gH/gL, HveA and HveC into a baculovirus expression system and/or mammalian cells and have expressed and obtained purified proteins.
In Aim 1, they will use these proteins to solve the structure of gD alone and/or in combination with receptor and/or antibody by X-ray diffraction analysis. They will map antibody and receptor binding domains on gD and look for conformational changes in gD that occur as a result of receptor binding.
In Aim 2, they will use quantitative assays that present gD or its receptor in the context of a membrane and study the interaction of the other protein as a soluble form. One approach will be to bind HSV to an ELISA plate and then determine if added soluble receptor inhibits the infection of cells that are added in fluid phase. Such studies will indicate that they are evaluating receptor binding to a viable virus. In a second approach, they will bind virions to a biosensor chip and flow soluble receptor across the chip to measure receptor-virus interactions. A third approach will be to express the receptor in the murine leukemia virus (MuLV)-receptor pseudotype system and use these particles to study binding of gDt.
In Aim 3, they will look for interactions among soluble forms of gD, gB and gH/gL in the presence and absence of gD receptors. In addition, they will study a series of gD/gH hybrid molecules for their ability to function in HSV infection in place of gD or gH/gL. Finally, they will study mutant forms of HSV including those that no longer require HveA or HveC for entry.
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