Work in the Longnecker Laboratory will focus primarily on experiments proposed in Aim 1.1 In this subaim, we have proposed the hypothesis that EBV gH/gL binding to integrins is an important determinant of fusion activation. To investigate this hypothesis, we will determine the binding capacity of of wt gH/gL and our collection of gH/gL mutants to integrins and function in fusion. There is a total of 5 different mutants that we propose to test. The integrin binding and gH functional studies will be done multiple times to obtain statistically significant data set that will allow us to fully categorize the various mutants. These studies will provide a basis for the biophysical experiments proposed in Subaim 1.2. To begin, we investigate the binding of soluble integrins to cells expressing wt gH/gL and our gH/gL mutants. Previous studies have shown that soluble integrins are functional for triggering fusion and bind to cells expressing integrins. The same cell lines used in the earlier studies expressing the secreted integrins αvβ3, αvβ6, and αvβ8 fused to alkaline phosphatase have been kindly provided to us by Stephen Nishimura (UCSF) and will be used in our studies. The integrin αvβ3 is negative for gH/gL binding and nonfunctional whereas αvβ6 and αvβ8 are positive for integrin binding and function. Briefly, the soluble integrins will be purified as described and bound to CHO cells expressing gH/gL and the gH/gL mutants. Binding will be monitored using alkaline phosphatase. Surface expressed gH/gL will be monitoring using the gH/gL monoclonal antibody E1D1 as we have done extensively in the past. In addition, we plan to monitor wt gH/gL and mutant gH/gL binding to AGS cells. AGS are a gastric carcinoma cell line and have been used extensively as a model of epithelial EBV infection and fusion. For these studies, we will make soluble versions of our gH/gL mutants. Fortunately, we have already made a wt secreted variant of gH/gL that we have used extensively which works well. The mutant secreted gH/gL variants will be made identical to this construct in which the gH transmembrane domain has been replaced by a flag tag and stop codon. These experiments will be confirmatory of our gH/gL binding assays to specific integrins as done in the first series of experiments and allow testing of binding in a biologically relevant cell line. Binding to integrins will be monitored using antibodies against the flag tag. Finally, we will perform a comprehensive analysis of the fusion function of each of our gH/gL mutants using our established epithelial and B cell fusion assays. As indicated in above, we plan to test each mutant in multiple times to insure we have statistical significant data and a clear idea of the function of each of our gH/gL mutants. From these studies, a comprehensive picture of the integrin binding phenotype of each of our mutants and how this binding alters B cell or epithelial cell fusion will be obtained that will form a basis of the remainder of the experiments proposed in Aim 1 and those in Aim 2.
|Effective start/end date||7/1/15 → 6/30/17|
- Stanford University (61048128-117572 // 1R21AI119480-01)
- National Institute of Allergy and Infectious Diseases (61048128-117572 // 1R21AI119480-01)
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