Structural mechanism of membrane remodeling during herpesvirus nuclear egress

Statement Of Work (Appendix E) Title: Structural mechanism of membrane remodeling during herpesvirus nuclear egress I. Background Information The proposed research requires implementation of several technical procedures that my lab has substantial experience with and performs on a regular basis. In addition, the work requires handling of pseudorabies virus (PRV, a veterinary herpesvirus) using BSL2 guidelines. I have been studying PRV for over a decade and have a BSL2 facility dedicated to herpesvirus propagation. II. Services to be Performed 1) I will purify PRV nucleocapsids from infected cell nuclei. This material is non-infectious, and will be supplied to Dr. Heldwein’s lab for further analysis. 2) I will produce recombinant PRV that encode mutations in the coding sequences of viral genes and insertions of fluorescent protein coding sequences. These viruses will be examined for propagation and cell-cell spread kinetics using plaque assays and single-step growth curves. We will also perform imaging of living infected cells using fluorescence microscopes in our BSL2 facility designed for this purpose. Imaging will be used for analysis of virus assembly and egress.