Core C - Virus/Vector

CORE C – PROJECT SUMMARY/ABSTRACT Core C. Virus/Vector Core Eric Johannsen, Core Leader Core C (Virus Core) provides time and cost-efficient generation, validation, distribution, and archival storage of wild-type (WT) and mutant viruses needed by all 6 research groups of this Program Project for their proposed studies. The Specific Aims are: (1) to produce validated stocks of WT, mutant, and revertant Epstein-Barr virus (EBV) and Kaposi Sarcoma Herpesvirus (KSHV); (2) to produce validated stocks of infectious mouse papillomavirus (MmuPV1) and human papillomavirus (HPV) pseudoviruses; and (3) to produce validated lentivirus library stocks. Mutant EBV (for Projects 2, 3, and 4) or KSHV (for Project 2) will be generated in E. coli starting with appropriate BACs using the scarless En Passant method.1 We can also make mutants, when appropriate in eukaryotic cells using CRISPR/Cas9 editing. In addition to standard methods, we have established a bioinformatic pipeline to analyze next generation sequencing data of herpesvirus genomes to ensure there are no unintended mutations within their unique regions. Whenever necessary, phenotypes will be validated using trans-complementation or the construction of revertant EBV/KSHV genomes. Virus stocks of WT, mutant, and revertant variants of EBV or KSHV will be generated using a protocol developed by Dr. Sugden, titered, and stored for use by all three EBV groups. Papillomavirus virions and pseudovirions will be generated (for Project 1) by co-transfection of 293T cells with (i) the desired capsid protein-expression plasmids, and (ii) the desired viral or luciferase/GFP-encoding DNAs targeted for encapsidation using protocols previously published by the Lambert/Ahlquist laboratories. Lentivirus libraries will be packaged and tittered using standardized protocols for use in Projects 2 and 4. Lenti and retrovirus transduction vectors with different antibiotic resistance genes have been generated and will be provided to Projects to allow transduction of multiple genes into cells. In addition to increased cost efficiency and quality control, the existence of this Core facilitates the use of common virus stocks that facilitate the interpretation of complementary data generated among the various research groups to foster shared aims within the projects. All 6 research groups associated with the 4 projects will be served by this Core facility.