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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA022443-42
Application #
9679480
Study Section
Special Emphasis Panel (ZCA1)
Project Start
Project End
Budget Start
2019-05-01
Budget End
2020-04-30
Support Year
42
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Weng, Chao; Lee, Denis; Gelbmann, Christopher B et al. (2018) Human Cytomegalovirus Productively Replicates In Vitro in Undifferentiated Oral Epithelial Cells. J Virol 92:
Bristol, Jillian A; Djavadian, Reza; Albright, Emily R et al. (2018) A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection. PLoS Pathog 14:e1007179
Romero-Masters, James C; Ohashi, Makoto; Djavadian, Reza et al. (2018) An EBNA3C-deleted Epstein-Barr virus (EBV) mutant causes B-cell lymphomas with delayed onset in a cord blood-humanized mouse model. PLoS Pathog 14:e1007221
UmaƱa, Angie C; Iwahori, Satoko; Kalejta, Robert F (2018) Direct Substrate Identification with an Analog Sensitive (AS) Viral Cyclin-Dependent Kinase (v-Cdk). ACS Chem Biol 13:189-199
Meyers, Jordan M; Grace, Miranda; Uberoi, Aayushi et al. (2018) Inhibition of TGF-? and NOTCH Signaling by Cutaneous Papillomaviruses. Front Microbiol 9:389
Uberoi, Aayushi; Yoshida, Satoshi; Lambert, Paul F (2018) Development of an in vivo infection model to study Mouse papillomavirus-1 (MmuPV1). J Virol Methods 253:11-17
Djavadian, Reza; Hayes, Mitchell; Johannsen, Eric (2018) CAGE-seq analysis of Epstein-Barr virus lytic gene transcription: 3 kinetic classes from 2 mechanisms. PLoS Pathog 14:e1007114
Chakravorty, Adityarup; Sugden, Bill (2018) Long-distance communication: Looping of human papillomavirus genomes regulates expression of viral oncogenes. PLoS Biol 16:e3000062
Chiu, Ya-Fang; Sugden, Bill (2018) Plasmid Partitioning by Human Tumor Viruses. J Virol 92:
Shin, Myeong-Kyun; Payne, Susan N; Bilger, Andrea et al. (2018) Activating Mutations in Pik3caContribute to Anal Carcinogenesis in the Presence or Absence of HPV-16 Oncogenes. Clin Cancer Res :

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