Background: Base J regulates Pol II transcription in trypanosomatids and is synthesized in two steps involving DNA hydroxylation and glucosylation. Results: JGT catalyzes the transfer of glucose from uridine diphosphoglucose to hmU in DNA. Conclusion: JGT is the glucosyltransferase involved in base J synthesis. Significance: JGT is the first known glucosyltransferase that modifies eukaryotic DNA.

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 Tbb HpHbR 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 Tbb HpHbR binding enhances the cytotoxicity; however, in the absence of Tbb HpHbR, 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 Tbb HpHbR. We found that T. b. brucei killing by TLF-2 was reduced in Tbb HpHbR-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 Tbb HpHbR 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.

Background: Base J regulates Pol II transcription. Results: JBP1 and -2 stimulate the first step of base J synthesis: hydroxylation of thymidine. Conclusion: JBP are Fe 2ϩ /2-OG-dependent dioxygenases sensitive to physiologically relevant O 2 tensions. Significance: These results predict that JBPs can act as oxygen sensors regulating trypanosome gene expression and adaption to different host niches.

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 (G 305 TTASSDTAITLIPR 319) 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. Viral infections induce potent immune responses, an immunopathogenesis that can lead to severe complications with sepsis or leaky capillary syndromes and very high mortality and limited effective treatments, a true unmet clinical need. Sepsis has an associated risk of disseminated intravascular coagulation (DIC) with thrombosis, hemorrhage and shock 1-3. One such group of viruses with known severe complications are the gammaherpesviruses (GHV). The murine gammaherpesvirus-68 (MHV-68) is a widely used, well-controlled laboratory model of GHV host-pathogen interaction with genetic similarity to the human viruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) 4. Inflammatory vasculitic syndromes (IVS) are a group of rare, heterogeneous and devastating inflammatory conditions of the body's extensive system of blood vessels with increased morbidity, including sudden loss of vision, aneurysm, aortic arch syndrome, stroke, and associated increases in mortality 5-7. The etiology of many systemic vasculitides is currently unknown, with proposed mechanisms ranging from induction by fungal spores

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.

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.

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. In vivo, 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 in vivo 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 in vivo function of conserved gammaherpesvirus genetic elements. We identified here MHV68 mghv-miR-M1-7-5p as critical for in vivo infection and then validated host EWSR1 (Ewing sarcoma breakpoint region 1) as the predominant target for this miRNA. Using novel, target-specific shRNA-expressing viruses, we determined that EWSR1 repression in vivo was essential for germinal center B cell infection. These findings provide the first in vivo demonstration of the biological significance of repression of a specific host mRNA by a gammaherpesvirus miRNA. IMPORTANCE 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. In vivo, 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 in vivo relevance of repression of targets of gammaherpesvirus miRNAs remains highly elusive. Here, we identified a murine gammaherpesvirus miRNA as critical for in vivo infection and validated the host mRNA EWSR1 (Ewing sarcoma breakpoint region 1) as the predominant target for this miRNA. Using a novel technology, we demonstrated that repression of EWSR1 was essential for in vivo infection of the critical B cell reservoir. These findings provide the first in vivo demonstration of the significance of repression of a specific host mRNA by a gammaherpesvirus miRNA.

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.