Identification of ebola glycoprotein mutants that exhibit increased transduction efficiency

Previous studies in the lab identified a series of alanine substitutions in the mucin domain-deleted Ebola virus glycoprotein 1 (EBOVΔO GP1) that enhance EBOV GP pseudovirion transduction efficiency by about two fold above wild type glycoprotein (1). To determine if combining these alanine substitutions resulted in even higher transduction levels, a series of mutants were generated and assessed for their ability to deliver pseudovirions into the highly permissive cell line SNB-19. The mutant EBOV GP 1 V96A/V97A was used as the base construct on to which other substitutions were added. A total of 8 combinations of alanine substitutions were assessed and transduction of the mutants could be characterized by one of three patterns: 1) enhancement of transduction, 2) decrease in transduction, or 3) no change in transduction in comparison to wild type and the previous mutant combinations. The best combination enhanced transduction in SNB-19 cells eight fold over wild type EBOVΔO GP and contained the mutations 96A/V97A/T83A/E106A/N107A. Previous transduction studies suggested that EBOV ΔO GP pseudovirion transduction efficiency would need to increase by 8-10 fold for this viral glycoprotein to be of value for in vivo pseudovirion gene therapy delivery studies. Our mutant 96A/V97A/T83A/E106A/N107A affords that enhanced transduction efficiency thereby providing an additional viral glycoprotein that efficiently delivers transgenes in vivo. With the recent success crystallizing the Ebola virus glycoprotein (56), we were able to use 3D modeling programs to visualize the location of the alanine substitutions, and identify other residues located in a similar context to identify additional potential transduction-enhancing substitutions. These newly identified residues consisting of three single mutations and two new combinations of mutations were