Janet E. Mertz - US grants

The long-term objective of the proposed research is to understand at the molecular level the roles played by large T antigen in regulating late gene expression of SV40. During this grant, we will test the validity of a number of hypotheses relating to this general problem. Specifically, we will: (i) determine whether large T antigen is required for late strand RNA synthesis by looking for this RNA in cells infected with a mutant of SV40 that does not encode this protein; (ii) determine whether late strand RNA synthesis can occur in the absence of viral DNA replication by testing with appropriate restriction endonucleases whether replication-defective mutants that synthesize this RNA in monkey cells and Xenopus oocytes are truly unable to undergo even a single round of viral DNA replication; (iii) determine whether large T antigen directly enhances synthesis of late strand RNA via its interactions with promoter-regulatory region sequences on the viral genome by examining a variety of T antigen-defective and promoter-regulatory region mutants for their ability to make late strand RNA; (iv) determine by S1 mapping of the 5' ends of the RNAs made in WT- and tsA58-infected cells whether T antigen-induced changes occur during the lytic cycle in the transcription initiation sites used for late strand RNA synthesis; and (v) determine whether a late strand transcriptional "attenuator" exists in SV40 by looking by S1 mapping for the presence of small RNAs with appropriate 3' ends in (a) WT- and mutant-infected monkey cells at various times after infection; and (b) Xenopus oocytes injected with WT and mutant SV40 DNAs and, in later experiments, preparations of virus-coded proteins. These studies should help us to learn about mechanisms by which tumor antigens can alter gene expression in ways that may on occasion lead to the transformation of cells to an oncogenic state.

DESCRIPTION (Applicant's Description): The purpose of this program is to provide training in viral oncology at the postdoctoral level. Trainees undertake a program of laboratory research with one or more faculty members. There are no formal course requirements or examinations; however, postdoctoral trainees audit appropriate lecture and seminar courses in virology and oncology. In addition, they are expected to publish, study independently, regularly attend and speak at seminars, journal clubs, and research meetings, and learn about the research of other trainees and faculty. Weekly seminars in both Tumor Virology and Molecular Virology serve as a focus for the training program. All trainees regularly attend one or both of these seminars and present at least one research report on their work every year. Trainers also attend one or both of these seminars which, on occasion, include outside speakers and highlights of recently held conferences. The faculty associated with this program are affiliated with several different schools and departments. They carry out research on viruses infecting a variety of organisms and employ a wide range of techniques. This variety of techniques and organisms insures a broad experience to trainees on this grant. The progress of all trainees is monitored by frequent (usually weekly) consultation with one or more trainers, by the yearly presentation of research results in the weekly seminars, and by the requirement for written annual progress reports for additional years of support. Trainees must have either a Ph.D. in biochemistry, microbiology, virology, oncology, genetics, or related areas, or a D.V.M. or an M.D. with an interest in viral oncology. Trainees are selected for support on this grant from applications received by faculty in this program. All applicants are required to provide a C.V., publication list, three letters of recommendation, and a statement of the specific research training proposed. A committee composed of the Program Director and 2 or 3 other senior trainers selects the trainees from among these applicants. After the training period, which is usually 2-3 years, trainees are qualified to assume responsible research positions related to tumor virology in a variety of institutions. Facilities associated with this training program are well-equipped virology, molecular biology, biochemistry and biotechnology laboratories in the McArdle Laboratory for Cancer Research, the Institute for Molecular Virology, and the Departments of Biochemistry, Chemical Engineering, Medical Microbiology and Immunology, Ophthalmology and Visual Sciences, and Pathology and Laboratory Medicine.

The objectives are to understand the molecular level some of the transcriptional and post-transcriptional mechanisms that regulate expression of genes of simian virus 40 (SV40), herpes simplex virus type 1 (HSV-1), hepatitis B virus (HBV) and, eventually, other clinically relevant tumor viruses. The first specific aim is to determine the functions and mechanisms of action of action of the pre-mRNA processing enhancer (PPE) of the thymidine kinase gene of HSV, a human immunodeficiency virus (HIV) Rev-response-like element, and its HIV Rev- like cellular trans-acting factor, the heterogeneous nuclear-ribonuclear protein (hnRNP) L, in the processing and nuclear export of pre-mRNAs and their utility in the expression of complementary DNAs by (a) identifying the nuclear export of pre-mRNAs and their utility in the expression of complementary DNAs by (a) identifying the bases involved in PPE function, (b) determining the functions and mechanisms by which hnRNP L mediates intro-independent mRNA biogenesis via binding this PPE, (c) identifying additional PPE-like elements in other intronless genes, and (d) determining the generality of PPEs enabling efficient intron-independent gene expression in mammalian cells. The second and third specific aims are to determine the mechanisms by which members of the steroid/thyroid hormone receptor superfamily and their ligands affect expression and replication of SV40 of the steroid/thyroid hormone receptor superfamily and their ligands affect expression and replication of SV40 and HBV by (a) examining the effects on transcription and virion production of some nuclear receptors that bind promoters of these two viruses and the ligands for these receptors, and (b) determining the mechanisms by which specific nuclear receptors, their ligands, the SV40 encoded oncoprotein large T- antigen, and a cellular factor, DAP, regulated transcription from the SV40 major late promoter. A variety of biochemical, molecular, genetical, and cellular techniques will be used. The findings from these studies should not only increase our understanding of fundamental mechanisms involved in regulation of mRNA biogenesis in viruses and mammals, but may also lead to the development of novel drugs for the treatment of diseases caused by viruses and improved vectors for use in gene therapy and the manufacture of biologically useful proteins.

[unreadable] DESCRIPTION (provided by applicant): Epstein-Barr virus (EBV) is a human gamma herpesvirus that infects most people by adulthood. Infection is associated with infectious mononucleosis, Burkitt's and non-Hodgkin's lymphomas, Hodgkin's disease, nasopharyngeal carcinoma, oral hairy leukoplakia, and some gastric carcinomas. The long-term goals of this research are (i) to elucidate mechanisms controlling latency versus lytic replication of EBV, and (ii) to apply this knowledge toward the development of new therapies for treating some EBV-associated diseases. Dr. Mertz and her colleagues have recently identified a novel cis-acting element, ZV, and its trans-acting factor, ZEB1, that play central roles in regulating EBV's latent-lytic switch gene, BZLF1, during latency and its reactivation by inducers such as TGF-beta and anti-immunoglobulins. The specific aims are the following: (i) Determine importance of ZEB1 in regulating EBV's life cycle via binding the ZV element in the BZLF1 promoter by (a) determining effects of mutations in the ZV element on establishment of latency and reactivation to lytic replication, (b) confirming ZEB1 regulates expression of the BZLF1 promoter via binding the ZV element, (c) determining the effects of ZEB1's abundance on establishment and maintenance of latency and reactivation using cell lines inducible for expression of ZEB1, and (d) looking for correlations between presence of ZEB1 and consequences of infection by EBV by measuring the abundance of ZEB1 mRNA and protein in a variety of human B-lymphocytic and epithelial normal and EBV-associated tumor tissues; and (ii) Elucidate mechanisms by which effectors of cellular signaling pathways induce latency or reactivation of EBV in part via modulating ZEB1 by (a) determining the effects of the EBV-encoded protein LMP1 on ZEB1's abundance, activity, and effects on EBV's life cycle using cells inducible for expression of LMP1 and LMP1 mutants of EBV, and b) testing models for activation of expression of the BZLF1 gene via ZEB1 by looking for correlations between expression and changes in the abundance, cellular localization, DNA-binding activity, co-purifying co-regulators, and post-translational modifications of ZEB1 after activation of cells with anti-immunoglobulins and TGF-beta. These studies should confirm our hypothesis regarding the central roles of ZEB1 and the ZV element in regulating EBV's life cycle and, possibly, lead to novel therapies for immunization against EBV and treatment of some EBV-associated diseases. [unreadable] [unreadable] [unreadable]

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus virus that can infect both B lymphocytes and epithelial cells. Depending upon the cell type and its state of differentiation, infection by EBV leads to either lytic replication with cell death or a latent infection. Latent EBV infections are associated with several B-cell and epithelial-cell malignancies including Burkitt's lymphoma, Hodgkin's disease, non-Hodgkin's lymphomas in immunocompromised individuals, nasopharyngeal carcinoma, and some gastric carcinomas. Thus, understanding regulation of EBV latency versus lytic replication is crucial for developing therapies to treat these diseases. The BZLF1 and BRLF1 genes of EBV encode multifunctional proteins essential for lytic replication. While the BZLF1 and BRLF1 promoters are inactive in latently infected cells, cell differentiation and activators of certain cell signaling pathways can induce BZLF1 and BRLF1 expression, promoting EBV reactivation into lytic replication. In this Project, we will (i) identify cellular factors that play key roles in regulating expression of these two immediate-early genes in both B cells and epithelial cells, and (ii) examine how these factors contribute to controlling the balance between latent and lytic EBV infection in a variety of cell types and during cellular differentiation. In conjunction with Project 5, these results may lead to new therapies for treating EBV-associated cancers.

Epstein-Barr virus (EBV) is a human herpesvirus that infects both B lymphocytes and epithelial cells. Depending upon cell type and differentiation state, infection leads to lytic replication with cell death or latent infection. Latent infections are associated with several epithelial- and B-cell malignancies. By identifying key cellular factors and pathways regulating EBV latency and lytic replication and drugs that affect their activities, we hope to develop lytic induction therapies for treating patients with EBV-associated cancers. The immediate- early BZLF1 and BRLF1 genes of EBV encode multifunctional proteins essential for lytic replication. While transcription from their promoters, Zp and Rp, respectively, is strongly silenced in latently infected cells, cell differentiation and factors activated by signaling pathways can induce their expression, leading to reactivation into lytic replication. We have identifled several physiologically relevant factors that play key roles in regulating this switch: ZEB1, ZEB2, p53, Oct-1, Oct-2, Smads via TGF-p signaling, and the Zp ZIIR element. We have preliminary data indicating important roles as well for HIF-1 a, Pax5, and Blimp-la and have identified 2 drugs, nufiin-3 and L-mimosine, that induce EBV reactivation via effects on some of these factors. We have developed a mouse model with a humanized immune system that can be use to examine factors affecting EBV-induced tumorigenesis and effects of drugs on growth and killing of EBV+ lymphomas. We have also identified a non-tumorigenic epithelial cell line, NOKs, in which we can study effects of differentiation state on EBV's life cycle. Here, we propose to determine: (i) how HIF-1 a, the ZEBs, PAX5, and Blimp-la play key roles in controlling EBV's latent-lytic balance in cells in culture, during differentiation of EBV+ NOKs and B cells, and in humanized mice; and (ii) whether nufiin-3 or L-mimosine together with HDAC inhibitors can efficiently reactivate EBV, leading to highly selective killing of some types of EBV+ cells lines in culture and tumors in humanized mice, especially when used in combination with ganciclovir, a nucleoside analog prodrug activated by a virus-encoded kinase. The findings obtained from these experiments will identify key cellular factors and pathways that regulate the balance between latency and lytic replication of EBV in epithelial and B cells, may identify effective lytic-inducing drugs for treating EBV-associated cancers, and may lead to a candidate vaccine for immunization against EBV by identifying an EBV mutant unable to establish viral latency. Drs. Kenney and Mertz collaborate extensively, including holding bimonthly joint research group meetings and co-authoring publications in which both labs made key contributions.

The purpose of this Core C (Virus Core) is to provide a time- and cost-efficient, quality-controlled mechanism by which all 7 research groups associated with this Program Project will have ready access to high-titer stocks of the wild-type (WT) and mutant viruses they will need for their proposed studies. The Specific Aims are: (1) to produce high-titer stocks of WT and mutant Epstein-Barr viruses (EBVs); (2) to produce high-titer stocks of WT and mutant human cytomegaloviruses (HCMVs); (3) to produce stocks of WT and mutant hepatitis B viruses (HBVs); and (4) to produce stocks of infectious mouse papillomavirus (MusPV) and human papillomavirus (HPV) pseudoviruses. Mutant EBVs (for Projects 3, 4, and 5) will be generated in E. co//starting with appropriate BACs using the markeriess two-step red-mediated recombination protocol of Tischer ef al. (2006). In addition to standard methods, the correctness of EBV mutant variants will be confirmed by the use of high throughput DNA sequencing to circumvent, hopefully, the need to generate and characterize wild-type reverts of them. High-titer virus stocks of WT and mutant variants of EBV will be generated using a protocol developed by Dr. Sugden, titered, and stored for use by all three EBV groups. WT and mutant variants of HCMV (for Project 3) will be generated starting with appropriate BACs also using the Tischer et al. protocols; the WT and mutant HCMVs will be plaque-purified, grown into high-titer virus stocks, and the titers ofthe stocks determined in primary human fibroblasts by standard methods. WT, infectious HBV virions will be produced (for Project 2) from HepAD38 cells, a HepG2 derivative that is stably transfected with a tetracycline-repressed (tet-off), inducible HBV expression system. HBV mutants will be constructed by recombinant DNA approaches in the on-P 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 will also result in 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 7 research groups associated with the 5 projects will be served by this Core facility as needed. It will be the first and only one of its type on the UW-Madison campus.

PROJECT 4 ? PROJECT SUMMARY/ABSTRACT Epstein-Barr virus (EBV) is associated with 2% of human cancers, including a variety of B-cell lymphomas, nasopharyngeal carcinomas, and some gastric cancers. Over the current funding period, we developed a new epithelial cell line infected with EBV that can be used to identify factors that regulate the switch from latency to lytic infection that occurs during differentiation of epithelial cells. Using this and other EBV-infected cell lines, we identified multiple cellular factors that play key roles in determining whether EBV remains dormant or reactivates into lytic replication. We also discovered three novel classes of drugs (iron chelators, NEDDylation inhibitors, and immunomodulatory drugs such as pomalidomide) that induce reactivation of latent EBV into lytic replication, two of which are already FDA-approved for other uses. Based upon our findings, we hypothesize that p53 family members and NEDDylation are key contributors to regulation of the EBV latent-to-lytic switch. Here, we propose to test this hypothesis by determining: (i) the roles played by p53 (the most commonly mutated protein in cancers) and its close family member, ?Np63, in regulating EBV?s life cycle during differentiation of EBV-infected epithelial cells and treatment of these cells with drugs that induce lytic EBV replication; and (ii) how the NEDDylation inhibitor, MLN4924, and the iron chelator, deferoxamine, induce lytic EBV replication in cells latently infected with this virus. The information obtained from these experiments will then be used to identify optimal combinations of these drugs, together with other FDA- approved drugs, for efficiently inducing EBV into lytic infection in cancer cells in which it is latent. We are hopeful that these studies will lead to new lytic-induction therapies for treating patients with EBV-associated cancers.

ABSTRACT This conference grant application requests funds to help support the 2018 International Conference on EBV & KSHV and the 2018 International Conference on DNA Tumor Viruses. The latter conference is focused on the molecular biology of papillomaviruses, polyomaviruses and adenoviruses. The two conferences will be held at the same venue (University of Wisconsin-Madison) and will partially overlap with joint sessions and common plenary speakers. This joint conference is unique and innovative in that it will be the first time all three of these conferences concerned with oncogenic DNA viruses (i.e., the International Workshop on Kaposi's Sarcoma-Associated Herpesvirus (KSHV) and Related Agents, the International Symposium on Epstein-Barr Virus (EBV) and Associated Diseases, and the DNA Tumor Viruses Meeting) will be held at a single site, the Memorial Union on the University of Wisconsin (UW) campus in Madison, WI. The funds will be used to reduce the conference registration costs for outstanding US pre- and post-doctoral trainees, recently independent investigators, historically under-represented minorities, and scientists from poor countries who study oncogenic DNA viruses. The goals of these meetings are consistent with the mission statements of the NIH, NCI, NIAID, and NIDCR, namely, to advance and promote the pace of research on infections associated with human cancer and other diseases, including in the setting of HIV-AIDS. These conferences will be hosted by the University of Wisconsin School of Medicine and Public Health. The joint EBV & KSHV Meeting will be held July 28th - August 1st, 2018 while the overlapping DNA Tumor Viruses Meeting will be held July 31st ? August 4th, 2018. All remaining costs for these conferences will be raised from registration fees paid by the conferees and contributions from host institutions, foundations, and pharmaceutical and biotechnology companies. The main focus of the EBV & KSHV meeting is on the biology of oncogenic herpesviruses and associated human diseases, with specific emphasis on viral pathogenesis, viral latency and reactivation, viral gene expression and replication, host responses to infection, epidemiology, vaccine development, and therapeutic intervention. In addition to EBV and KSHV, studies related to herpesvirus saimiri (HVS), murine herpesvirus-68 (MHV-68), rhesus rhadinovirus (RRV), and rhesus lymphocryptovirus (rhLCV) will be presented. The main focus of the DNA Tumor Viruses Meeting is on similar topic areas, but as they relate to the biology and diseases associated with human, primate, and other mammalian papilloma, polyoma, and adenoviruses. The session topics held on August 1st will be ones where there exists overlap of pathways used by these various classes of oncogenic viruses (e.g., viral genome expression and replication, co-infections, virus-host interactions) to optimize the potential for synergistic interactions among the conferees and the potential for new collaborations.