Publications by authors named "Whitney Bullard"

11 Publications

  • Page 1 of 1

EBV miRNAs are potent effectors of tumor cell transcriptome remodeling in promoting immune escape.

PLoS Pathog 2021 May 6;17(5):e1009217. Epub 2021 May 6.

Department of Pathology, Tulane University School of Medicine, Tulane Cancer Center, New Orleans, Louisiana, United States of America.

The Epstein Barr virus (EBV) contributes to the tumor phenotype through a limited set of primarily non-coding viral RNAs, including 31 mature miRNAs. Here we investigated the impact of EBV miRNAs on remodeling the tumor cell transcriptome. Strikingly, EBV miRNAs displayed exceptionally abundant expression in primary EBV-associated Burkitt's Lymphomas (BLs) and Gastric Carcinomas (GCs). To investigate viral miRNA targeting, we used the high-resolution approach, CLASH in GC and BL cell models. Affinity constant calculations of targeting efficacies for CLASH hits showed that viral miRNAs bind their targets more effectively than their host counterparts, as did Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) miRNAs. Using public BL and GC RNA-seq datasets, we found that high EBV miRNA targeting efficacies translates to enhanced reduction of target expression. Pathway analysis of high efficacy EBV miRNA targets showed enrichment for innate and adaptive immune responses. Inhibition of the immune response by EBV miRNAs was functionally validated in vivo through the finding of inverse correlations between EBV miRNAs and immune cell infiltration and T-cell diversity in BL and GC datasets. Together, this study demonstrates that EBV miRNAs are potent effectors of the tumor transcriptome that play a role in suppressing host immune response.
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http://dx.doi.org/10.1371/journal.ppat.1009217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8130916PMC
May 2021

Immune protection is dependent on the gut microbiome in a lethal mouse gammaherpesviral infection.

Sci Rep 2020 02 11;10(1):2371. Epub 2020 Feb 11.

Center for Personalized Diagnostics, The Biodesign Institute, Arizona State University, Tempe, Arizona, USA.

Immunopathogenesis in systemic viral infections can induce a septic state with leaky capillary syndrome, disseminated coagulopathy, and high mortality with limited treatment options. Murine gammaherpesvirus-68 (MHV-68) intraperitoneal infection is a gammaherpesvirus model for producing severe vasculitis, colitis and lethal hemorrhagic pneumonia in interferon gamma receptor-deficient (IFNγR) mice. In prior work, treatment with myxomavirus-derived Serp-1 or a derivative peptide S-7 (GTTASSDTAITLIPR) induced immune protection, reduced disease severity and improved survival after MHV-68 infection. Here, we investigate the gut bacterial microbiome in MHV-68 infection. Antibiotic suppression markedly accelerated MHV-68 pathology causing pulmonary consolidation and hemorrhage, increased mortality and specific modification of gut microbiota. Serp-1 and S-7 reduced pulmonary pathology and detectable MHV-68 with increased CD3 and CD8 cells. Treatment efficacy was lost after antibiotic treatments with associated specific changes in the gut bacterial microbiota. In summary, transkingdom host-virus-microbiome interactions in gammaherpesvirus infection influences gammaherpesviral infection severity and reduces immune modulating therapeutic efficacy.
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http://dx.doi.org/10.1038/s41598-020-59269-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012916PMC
February 2020

Identification of murine gammaherpesvirus 68 miRNA-mRNA hybrids reveals miRNA target conservation among gammaherpesviruses including host translation and protein modification machinery.

PLoS Pathog 2019 08 8;15(8):e1007843. Epub 2019 Aug 8.

Dept. of Molecular Genetics and Microbiology, UF Health Cancer Center, University of Florida, Gainesville, Florida, United States of America.

Gammaherpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), establish lifelong latent infection in B cells and are associated with a variety of tumors. In addition to protein coding genes, these viruses encode numerous microRNAs (miRNAs) within their genomes. While putative host targets of EBV and KSHV miRNAs have been previously identified, the specific functions of these miRNAs during in vivo infection are largely unknown. Murine gammaherpesvirus 68 (MHV68) is a natural pathogen of rodents that is genetically related to both EBV and KSHV, and thus serves as an excellent model for the study of EBV and KSHV genetic elements such as miRNAs in the context of infection and disease. However, the specific targets of MHV68 miRNAs remain completely unknown. Using a technique known as qCLASH (quick crosslinking, ligation, and sequencing of hybrids), we have now identified thousands of Ago-associated, direct miRNA-mRNA interactions during lytic infection, latent infection and reactivation from latency. Validating this approach, detailed molecular analyses of specific interactions demonstrated repression of numerous host mRNA targets of MHV68 miRNAs, including Arid1a, Ctsl, Ifitm3 and Phc3. Notably, of the 1,505 MHV68 miRNA-host mRNA targets identified in B cells, 86% were shared with either EBV or KSHV, and 64% were shared among all three viruses, demonstrating significant conservation of gammaherpesvirus miRNA targeting. Pathway analysis of MHV68 miRNA targets further revealed enrichment of cellular pathways involved in protein synthesis and protein modification, including eIF2 Signaling, mTOR signaling and protein ubiquitination, pathways also enriched for targets of EBV and KSHV miRNAs. These findings provide substantial new information about specific targets of MHV68 miRNAs and shed important light on likely conserved functions of gammaherpesvirus miRNAs.
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http://dx.doi.org/10.1371/journal.ppat.1007843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6687095PMC
August 2019

A Gammaherpesvirus MicroRNA Targets EWSR1 (Ewing Sarcoma Breakpoint Region 1) To Promote Latent Infection of Germinal Center B Cells.

mBio 2019 07 30;10(4). Epub 2019 Jul 30.

Department of Molecular Genetics and Microbiology, UF Health Cancer Center, College of Medicine, University of Florida, Gainesville, Florida, USA

Gammaherpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), directly contribute to the genesis of multiple types of malignancies, including B cell lymphomas. , these viruses infect B cells and manipulate B cell biology to establish lifelong latent infection. To accomplish this, gammaherpesviruses employ an array of gene products, including microRNAs (miRNAs). Although numerous host mRNA targets of gammaherpesvirus miRNAs have been identified, the relevance of repression of these targets remains elusive due to species restriction. Murine gammaherpesvirus 68 (MHV68) provides a robust virus-host system to dissect the function of conserved gammaherpesvirus genetic elements. We identified here MHV68 - as critical for infection and then validated host (Ewing sarcoma breakpoint region 1) as the predominant target for this miRNA. Using novel, target-specific shRNA-expressing viruses, we determined that repression was essential for germinal center B cell infection. These findings provide the first demonstration of the biological significance of repression of a specific host mRNA by a gammaherpesvirus miRNA. Gammaherpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), directly contribute to the genesis of multiple types of malignancies. , these viruses infect B cells and manipulate B cell biology to establish lifelong infection. To accomplish this, gammaherpesviruses employ an array of gene products, including miRNAs, short noncoding RNAs that bind to and repress protein synthesis from specific target mRNAs. The relevance of repression of targets of gammaherpesvirus miRNAs remains highly elusive. Here, we identified a murine gammaherpesvirus miRNA as critical for infection and validated the host mRNA (Ewing sarcoma breakpoint region 1) as the predominant target for this miRNA. Using a novel technology, we demonstrated that repression of was essential for infection of the critical B cell reservoir. These findings provide the first demonstration of the significance of repression of a specific host mRNA by a gammaherpesvirus miRNA.
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http://dx.doi.org/10.1128/mBio.00996-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667617PMC
July 2019

Connivance, Complicity, or Collusion? The Role of Noncoding RNAs in Promoting Gammaherpesvirus Tumorigenesis.

Trends Cancer 2018 11 10;4(11):729-740. Epub 2018 Oct 10.

Department of Molecular Genetics and Microbiology, UF Health Cancer Center, University of Florida, Gainesville, FL, USA. Electronic address:

EBV and KSHV are etiologic agents of multiple types of lymphomas and carcinomas. The frequency of EBV or KSHV malignancies arising in immunocompromised individuals reflects the intricate evolutionary balance established between these viruses and their immunocompetent hosts. However, the specific mechanisms by which these pathogens drive tumorigenesis remain poorly understood. In recent years an enormous array of cellular and viral noncoding RNAs (ncRNAs) have been discovered, and host ncRNAs have been revealed as contributory factors to every single cancer hallmark cellular process. As new evidence emerges that gammaherpesvirus ncRNAs also alter host processes and viral factors dysregulate host ncRNA expression, and as novel viral ncRNAs continue to be discovered, we examine the contribution of small, non-miRNA ncRNAs and long ncRNAs to gammaherpesvirus tumorigenesis.
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http://dx.doi.org/10.1016/j.trecan.2018.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317380PMC
November 2018

Mouse Gamma Herpesvirus MHV-68 Induces Severe Gastrointestinal (GI) Dilatation in Interferon Gamma Receptor-Deficient Mice (IFNγR) That Is Blocked by Interleukin-10.

Viruses 2018 09 23;10(10). Epub 2018 Sep 23.

Department of Medicine, Divisions of Cardiovascular Medicine and Rheumatology, University of Florida, Gainesville, FL 32610-0277, USA.

Inflammatory bowel disease (IBD) and infection cause gastrointestinal (GI) distension and, in severe cases, toxic megacolon with risk of perforation and death. Herpesviruses have been linked to severe GI dilatation. MHV-68 is a model for human gamma herpesvirus infection inducing GI dilatation in interleukin-10 (IL-10)-deficient mice but is benign in wildtype mice. MHV-68 also causes lethal vasculitis and pulmonary hemorrhage in interferon gamma receptor-deficient (IFNγR) mice, but GI dilatation has not been reported. In prior work the Myxomavirus-derived anti-inflammatory serpin, Serp-1, improved survival, reducing vasculitis and pulmonary hemorrhage in MHV-68-infected IFNγR mice with significantly increased IL-10. IL-10 has been investigated as treatment for GI dilatation with variable efficacy. We report here that MHV-68 infection produces severe GI dilatation with inflammation and gut wall degradation in 28% of INFγR mice. Macrophage invasion and smooth muscle degradation were accompanied by decreased concentrations of T helper (Th2), B, monocyte, and dendritic cells. Plasma and spleen IL-10 were significantly reduced in mice with GI dilatation, while interleukin-1 beta (IL-1β), IL-6, tumor necrosis factor alpha (TNFα) and INFγ increased. Treatment of gamma herpesvirus-infected mice with exogenous IL-10 prevents severe GI inflammation and dilatation, suggesting benefit for herpesvirus-induced dilatation.
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http://dx.doi.org/10.3390/v10100518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213885PMC
September 2018

A method for the efficient and selective identification of 5-hydroxymethyluracil in genomic DNA.

Biol Methods Protoc 2017 Jan 25;2(1). Epub 2017 Jan 25.

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA.

Recently, 5-hydroxymethyluracil (5hmU) was identified in mammalian genomic DNA as an oxidative product of thymine by the ten-eleven translocation (TET) proteins. While the biological role of this modification remains unclear, identifying its genomic location will assist in elucidating function. Here we present a rapid and robust method to selectively tag and enrich genomic regions containing 5hmU. This method involves the selective glucosylation of 5hmU residues by the base J glucosyltransferase from trypanosomes creating glucosylhydroxymethyluracil (base J). The base J can then be efficiently and selectively pulled down by antibodies against base J or by J-binding protein 1. DNA that is enriched is suitable for analysis by quantitative PCR or sequencing. We utilized this tagging reaction to provide proof of concept for the enrichment of 5hmU containing DNA from a pool that contains modified and unmodified DNA. Furthermore, we demonstrate that the base J pull-down assay identifies 5hmU at specific regions of the trypanosome genome involved in transcriptional repression. The method described here will allow for a greater understanding of the functional role and dynamics of 5hmU in biology.
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http://dx.doi.org/10.1093/biomethods/bpw006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5741180PMC
January 2017

Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA.

Mol Biochem Parasitol 2015 Dec 24;204(2):77-80. Epub 2016 Jan 24.

From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, United States. Electronic address:

Telomeric DNA of trypanosomatids possesses a modified thymine base, called base J, that is synthesized in a two-step process; the base is hydroxylated by a thymidine hydroxylase forming hydroxymethyluracil (hmU) and a glucose moiety is then attached by the J-associated glucosyltransferase (JGT). To examine the importance of JGT in modifiying specific thymine in DNA, we used a Leishmania episome system to demonstrate that the telomeric repeat (GGGTTA) stimulates J synthesis in vivo while mutant telomeric sequences (GGGTTT, GGGATT, and GGGAAA) do not. Utilizing an in vitro GT assay we find that JGT can glycosylate hmU within any sequence with no significant change in Km or kcat, even mutant telomeric sequences that are unable to be J-modified in vivo. The data suggests that JGT possesses no DNA sequence specificity in vitro, lending support to the hypothesis that the specificity of base J synthesis is not at the level of the JGT reaction.
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http://dx.doi.org/10.1016/j.molbiopara.2016.01.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889113PMC
December 2015

Identification of the glucosyltransferase that converts hydroxymethyluracil to base J in the trypanosomatid genome.

J Biol Chem 2014 Jul 2;289(29):20273-82. Epub 2014 Jun 2.

From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602 and

O-linked glucosylation of thymine in DNA (base J) is an important regulatory epigenetic mark in trypanosomatids. β-d-glucopyranosyloxymethyluracil (base J) synthesis is initiated by the JBP1/2 enzymes that hydroxylate thymine, forming 5-hydroxymethyluracil (hmU). hmU is then glucosylated by a previously unknown glucosyltransferase. A recent computational screen identified a possible candidate for the base J-associated glucosyltransferase (JGT) in trypanosomatid genomes. We demonstrate that recombinant JGT utilizes uridine diphosphoglucose to transfer glucose to hmU in the context of dsDNA. Mutation of conserved residues typically involved in glucosyltransferase catalysis impairs DNA glucosylation in vitro. The deletion of both alleles of JGT from the genome of Trypanosoma brucei generates a cell line that completely lacks base J. Reintroduction of JGT in the JGT KO restores J synthesis. Ablation of JGT mRNA levels by RNAi leads to the sequential reduction in base J and increased levels of hmU that dissipate rapidly. The analysis of JGT function confirms the two-step J synthesis model and demonstrates that JGT is the only glucosyltransferase enzyme required for the second step of the pathway. Similar to the activity of the related Ten-Eleven Translocation (TET) family of dioxygenases on 5mC, our studies also suggest the ability of the base J-binding protein enzymes to catalyze iterative oxidation of thymine in trypanosome DNA. Here we discuss the regulation of hmU and base J formation in the trypanosome genome by JGT and base J-binding protein.
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http://dx.doi.org/10.1074/jbc.M114.579821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106341PMC
July 2014

JBP1 and JBP2 proteins are Fe2+/2-oxoglutarate-dependent dioxygenases regulating hydroxylation of thymidine residues in trypanosome DNA.

J Biol Chem 2012 Jun 18;287(24):19886-95. Epub 2012 Apr 18.

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229, USA.

We have recently demonstrated that O-linked glucosylation of thymine in trypanosome DNA (base J) regulates polymerase II transcription initiation. In vivo analysis has indicated that base J synthesis is initiated by the hydroxylation of thymidine by proteins (JBP1 and JBP2) homologous to the Fe(2+)/2-oxoglutarate (2-OG)-dependent dioxygenase superfamily where hydroxylation is driven by the oxidative decarboxylation of 2-OG, forming succinate and CO(2). However, no direct evidence for hydroxylase activity has been reported for the JBP proteins. We now demonstrate recombinant JBP1 hydroxylates thymine specifically in the context of dsDNA in a Fe(2+)-, 2-OG-, and O(2)-dependent manner. Under anaerobic conditions, the addition of Fe(2+) to JBP1/2-OG results in the formation of a broad absorption spectrum centered at 530 nm attributed to metal chelation of 2-OG bound to JBP, a spectroscopic signature of Fe(2+)/2-OG-dependent dioxygenases. The N-terminal thymidine hydroxylase domain of JBP1 is sufficient for full activity and mutation of residues involved in coordinating Fe(2+) inhibit iron binding and thymidine hydroxylation. Hydroxylation in vitro and J synthesis in vivo is inhibited by known inhibitors of Fe(2+)/2-OG-dependent dioxygenases. The data clearly demonstrate the JBP enzymes are dioxygenases acting directly on dsDNA, confirming the two-step J synthesis model. Growth of trypanosomes in hypoxic conditions decreases JBP1 and -2 activity, resulting in reduced levels of J and changes in parasite virulence previously characterized in the JBP KO. The influence of environment upon J biosynthesis via oxygen-sensitive regulation of JBP1/2 has exciting implications for the regulation of gene expression and parasite adaptation to different host niches.
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http://dx.doi.org/10.1074/jbc.M112.341974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370173PMC
June 2012

Haptoglobin-hemoglobin receptor independent killing of African trypanosomes by human serum and trypanosome lytic factors.

Virulence 2012 Jan-Feb;3(1):72-6. Epub 2012 Jan 1.

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.

The haptoglobin-hemoglobin receptor (HpHbR) of African trypanosomes plays a critical role in human innate immunity against these parasites. Localized to the flagellar pocket of the veterinary pathogen Trypanosoma brucei brucei this receptor binds Trypanosome Lytic Factor-1 (TLF-1), a subclass of human high-density lipoprotein (HDL) facilitating endocytosis, lysosomal trafficking and subsequent killing. Recently, we found that group 1 Trypanosoma brucei gambiense does not express a functional HpHbR. We now show that loss of the TbbHpHbR reduces the susceptibility of T. b. brucei to human serum and TLF-1 by 100- and 10,000-fold, respectively. The relatively high concentrations of human serum and TLF-1 needed to kill trypanosomes lacking the HpHbR indicates that high affinity TbbHpHbR binding enhances the cytotoxicity; however, in the absence of TbbHpHbR, other receptors or fluid phase endocytosis are sufficient to provide some level of susceptibility. Human serum contains a second innate immune factor, TLF-2, that has been suggested to kill trypanosomes independently of the TbbHpHbR. We found that T. b. brucei killing by TLF-2 was reduced in TbbHpHbR-deficient cells but to a lesser extent than TLF-1. This suggests that both TLF-1 and TLF-2 can be taken up via the TbbHpHbR but that alternative pathways exist for the uptake of these toxins. Together the findings reported here extend our previously published studies and suggest that group 1 T. b. gambiense has evolved multiple mechanisms to avoid killing by trypanolytic human serum factors.
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http://dx.doi.org/10.4161/viru.3.1.18295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3337153PMC
September 2012