The broad, long term objectives of this project are to understand the control of human herpesvirus replication and reactivation from latency. Epstein Barr virus (EBV) is a ubiquitous herpesvirus associated with human lymphoid and epithelial malignancies. Lytic replication and expression of lytic gene products during virus reactivation play an important role in pathogenesis. The focus of this application is to investigate the mechanisms of action of SM, an EBV protein that is expressed during lytic replication and is essential for production of infectious virus. SM binds to RNA and enhances expression of several EBV lytic genes including those critical for capsid formation and infectivity. We have shown that these effects are gene-specific and that SM is preferentially required for expression of a subset of EBV genes. A fundamental question is how SM specifically facilitates expression of the mRNAs of these target genes. While SM stabilizes some target RNAs, and has long been thought to only act post-transcriptionally, we provide new evidence that it also facilitates EBV gene transcription. Identifying the targets of these separate mechanisms will allow a molecular dissection of each function. A high throughput screening assay to identify SM inhibitors showed that spironolactone (SPR), a clinically approved mineralocorticoid-blocking agent, has potent anti-SM and antiviral properties. We have found that SPR acts to destabilize a cellular transcription factor, XPB which appears to be uniquely involved in EBV gene transcription. SPR is therefore hypothesized to act through effects on host cell transcription factors that are necessary for SM function. The project has three specific aims. The first is to determine whether SM facilitates transcription initiation/elongation or RNA stabilization to achieve its effects on each of its specific targets. We hypothesize that the combined effect of SM on transcription and mRNA stability of individual genes varies based on specific target gene characteristics. By defining which promoters SM affects, and which RNAs it stabilizes, the mechanisms of transcriptional and post-transcriptional enhancement will be separated and individually investigated.
The second aim i s to investigate the mechanisms by which spironolactone (SPR) inhibits SM function and to expand its utility as an antiviral agent. Using a synthetic chemistry approach, the mineralocorticoid blocking activity will be ablated from SPR, to demonstrate that it can be separated from antiviral activity. SPR derivatives will also be made to identify its target proteins that will then be identified by proteomic techniques. In the third aim, the unique role of XPB in EBV transcriptional initiation and elongation and its cooperation with SM will be investigated. The combination of these three aims will not only clarify the mechanism of action of SM but will lay the basis for targeting unique interaction points of viral and cellular gene expression for drug development.

Public Health Relevance

ThisproposalistostudygrowthofEpsteinBarrvirus,orEBV,avirusthatisassociatedwithcancer.The projectwillstudyaproteinknowasEBVSMthatisessentialforproducinginfectiousvirus.Theprojectis alsoaimedatdevelopingdrugsthatcanblockreplicationbyinhibitingthisprotein.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Virology - A Study Section (VIRA)
Program Officer
Daschner, Phillip J
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University of Utah
Internal Medicine/Medicine
Schools of Medicine
Salt Lake City
United States
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Church, Trenton Mel; Verma, Dinesh; Thompson, Jacob et al. (2018) Efficient Translation of Epstein-Barr Virus (EBV) DNA Polymerase Contributes to the Enhanced Lytic Replication Phenotype of M81 EBV. J Virol 92:
Swaminathan, Sankar; Schlaberg, Robert; Lewis, Julia et al. (2016) Fatal Zika Virus Infection with Secondary Nonsexual Transmission. N Engl J Med 375:1907-1909
Verma, Dinesh; Thompson, Jacob; Swaminathan, Sankar (2016) Spironolactone blocks Epstein-Barr virus production by inhibiting EBV SM protein function. Proc Natl Acad Sci U S A 113:3609-14
Thompson, Jacob; Verma, Dinesh; Li, DaJiang et al. (2016) Identification and Characterization of the Physiological Gene Targets of the Essential Lytic Replicative Epstein-Barr Virus SM Protein. J Virol 90:1206-21
Verma, Dinesh; Li, Da-Jiang; Krueger, Brian et al. (2015) Identification of the physiological gene targets of the essential lytic replicative Kaposi's sarcoma-associated herpesvirus ORF57 protein. J Virol 89:1688-702
Li, Da-Jiang; Verma, Dinesh; Mosbruger, Tim et al. (2014) CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription. PLoS Pathog 10:e1003880
Verma, Dinesh; Kim, Eun A; Swaminathan, Sankar (2013) Cell-based screening assay for antiviral compounds targeting the ability of herpesvirus posttranscriptional regulatory proteins to stabilize viral mRNAs. J Virol 87:10742-51
Li, Da-Jiang; Verma, Dinesh; Swaminathan, Sankar (2012) Binding of cellular export factor REF/Aly by Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is not required for efficient KSHV lytic replication. J Virol 86:9866-74
Verma, Dinesh; Bais, Swarna; Gaillard, Melusine et al. (2010) Epstein-Barr Virus SM protein utilizes cellular splicing factor SRp20 to mediate alternative splicing. J Virol 84:11781-9
Han, Zhao; Verma, Dinesh; Hilscher, Chelsey et al. (2009) General and target-specific RNA binding properties of Epstein-Barr virus SM posttranscriptional regulatory protein. J Virol 83:11635-44

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