The purpose of Core C (Virus Core) is to provide a time and cost efficient mechanism for the generation, validation, distribution, and archival storage of wild-type (WT) and mutant viruses needed by all 8 research groups associated with 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 WT and mutant hepatitis B viruses (HBVs); (3) to produce validated stocks of infectious mouse papillomavirus (MmuPV1) and human papillomavirus (HPV) pseudoviruses. (1). Mutant EBV (for Projects 3, 4, and 5) or KSHV (for Project 3) will be generated in E. coli starting with appropriate BACs using the scarless En Passant method 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 the unique regions. Whenever necessary, phenotypes will be validated using transcomplementation 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 four EBV groups. HBV virions will be produced (for Project 2) from HepAD38 cells, a HepG2 derivative that is stably transfected with a tetracycline-inducible HBV expression system. HBV mutants will be constructed by recombinant DNA approaches in vectors already developed by the Loeb laboratory. 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. In addition to increased cost efficiency and quality control, the existence of this Core facilitates the use of common virus stocks that will make the interpretation of complementary data generated among the various research groups more reliably merged toward achieving shared aims within the projects. All 8 research groups associated with the 5 projects will be served by this Core facility as needed.
The purpose of this Virus Core is to provide a time and cost efficient mechanism by which all eight research groups associated with this Program Project will have ready access to validated, high-titer stocks of the wild- type and mutant viruses that they require for their proposed studies.
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