Publications by authors named "Scott Tibbetts"

41 Publications

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

Epstein-Barr virus EBER1 and murine gammaherpesvirus TMER4 share conserved in vivo function to promote B cell egress and dissemination.

Proc Natl Acad Sci U S A 2019 12 3;116(51):25392-25394. Epub 2019 Dec 3.

Department of Molecular Genetics & Microbiology, University of Florida Health Cancer Center, University of Florida, Gainesville, FL 32610

The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, γHV68, MuHV-4) establish life-long latency in circulating B cells. The precise determinants that mediate in vivo gammaherpesvirus latency and tumorigenesis remain unclear. The EBV-encoded RNAs (EBERs) are among the first noncoding RNAs ever identified and have been the subject of decades of studies; however, their biological roles during in vivo infection remain unknown. Herein, we use a series of refined virus mutants to define the active isoform of MHV68 noncoding RNA TMER4 and demonstrate that EBV EBER1 functionally conserves this activity in vivo to promote egress of infected B cells from lymph nodes into peripheral circulation.
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http://dx.doi.org/10.1073/pnas.1915752116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926008PMC
December 2019

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

Genome-wide Transcript Structure Resolution Reveals Abundant Alternate Isoform Usage from Murine Gammaherpesvirus 68.

Cell Rep 2019 06;27(13):3988-4002.e5

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

The gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, MuHV-4, γHV68), are etiologic agents of a wide range of lymphomas and non-hematological malignancies. These viruses possess large and highly dense dsDNA genomes that feature >80 bidirectionally positioned open reading frames (ORFs). The abundance of overlapping transcripts and extensive splicing throughout these genomes have until now prohibited high throughput-based resolution of transcript structures. Here, we integrate the capabilities of long-read sequencing with the accuracy of short-read platforms to globally resolve MHV68 transcript structures using the transcript resolution through integration of multi-platform data (TRIMD) pipeline. This approach reveals highly complex features, including: (1) pervasive overlapping transcript structures; (2) transcripts containing intra-gene or trans-gene splices that yield chimeric ORFs; (3) antisense and intergenic transcripts containing ORFs; and (4) noncoding transcripts. This work sheds light on the underappreciated complexity of gammaherpesvirus transcription and provides an extensively revised annotation of the MHV68 transcriptome.
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http://dx.doi.org/10.1016/j.celrep.2019.05.086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071827PMC
June 2019

Gammaherpesvirus RNAs Come Full Circle.

mBio 2019 04 2;10(2). Epub 2019 Apr 2.

Department of Pathology, Tulane University School of Medicine, Tulane Cancer Center, New Orleans, Louisiana, USA

After an adaptive immune response is mounted, gammaherpesviruses achieve persistence through the utilization of viral noncoding RNAs to craft a suitable host cell environment in an immunologically transparent manner. While gammaherpesvirus long noncoding RNAs (lncRNAs) and microRNAs have been recognized for some time and have been actively investigated, a recent spate of reports have now identified repertoires of the circular RNA (circRNA) class of noncoding RNAs in both the lymphocryptovirus and rhadinovirus genera of gammaherpesviruses. Despite the recent nature of these findings, the detection of circRNAs across viruses and viral gene expression programs, the conservation of some viral circRNAs, and their detection in the clinical setting already raises the spectrum of functional importance in gammaherpesvirus biology and associated malignancies. Here, we provide an overview of currently known gammaherpesvirus circular RNAs and discuss reported physical and contextual properties that may be germane to future functional studies. With the Epstein-Barr virus (EBV) circRNAome being the most extensively studied to date, our discussions will be weighted toward EBV circRNAs while also addressing circRNAs discovered in the rhesus macaque lymphocryptovirus (rLCV), the Kaposi's sarcoma herpesvirus (KSHV), and the murid gammaherpesvirus 68 (MHV68). We hope that this will help set the stage for future investigations into the functions and relevance of this new class of viral noncoding RNAs in infection and disease.
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http://dx.doi.org/10.1128/mBio.00071-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445933PMC
April 2019

Gammaherpesvirus Readthrough Transcription Generates a Long Non-Coding RNA That Is Regulated by Antisense miRNAs and Correlates with Enhanced Lytic Replication In Vivo.

Noncoding RNA 2019 Jan 10;5(1). Epub 2019 Jan 10.

Department of Molecular Genetics & Microbiology, UF Health Cancer Center, University of Florida, Gainesville, Florida, 32610, USA.

Gammaherpesviruses, including the human pathogens Epstein⁻Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are oncogenic viruses that establish lifelong infections in hosts and are associated with the development of lymphoproliferative diseases and lymphomas. Recent studies have shown that the majority of the mammalian genome is transcribed and gives rise to numerous long non-coding RNAs (lncRNAs). Likewise, the large double-stranded DNA virus genomes of herpesviruses undergo pervasive transcription, including the expression of many as yet uncharacterized lncRNAs. Murine gammaperherpesvirus 68 (MHV68, MuHV-4, HV68) is a natural pathogen of rodents, and is genetically and pathogenically related to EBV and KSHV, providing a highly tractable model for studies of gammaherpesvirus biology and pathogenesis. Through the integrated use of parallel data sets from multiple sequencing platforms, we previously resolved transcripts throughout the MHV68 genome, including at least 144 novel transcript isoforms. Here, we sought to molecularly validate novel transcripts identified within the locus, which harbors genes that code for the chemokine binding protein M3, the latency B cell signaling protein M2, and 10 microRNAs (miRNAs). Using strand-specific northern blots, we validated the presence of a 3.91 kb polyadenylated transcript that initiates at the M3 transcription start site and reads through the M3 open reading frame (ORF), the M3 poly(a) signal sequence, and the M2 ORF. This unexpected transcript was solely localized to the nucleus, strongly suggesting that it is not translated and instead may function as a lncRNA. Use of an MHV68 mutant lacking two -antisense pre-miRNA stem loops resulted in highly increased expression of and increased virus replication in the lungs of infected mice, demonstrating a key role for these RNAs in regulation of lytic infection. Together these findings suggest the possibility of a tripartite regulatory relationship between the lncRNA , antisense miRNAs, and the latency gene .
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http://dx.doi.org/10.3390/ncrna5010006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468771PMC
January 2019

Comparative Analysis of Gammaherpesvirus Circular RNA Repertoires: Conserved and Unique Viral Circular RNAs.

J Virol 2019 03 5;93(6). Epub 2019 Mar 5.

Department of Pathology, Tulane University School of Medicine, Tulane Cancer Center, New Orleans, Louisiana, USA

Recent studies have identified circular RNAs (circRNAs) expressed from the Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) human DNA tumor viruses. To gain initial insights into the potential relevance of EBV circRNAs in virus biology and disease, we assessed the circRNAome of the interspecies homologue rhesus macaque lymphocryptovirus (rLCV) in a naturally occurring lymphoma from a simian immunodeficiency virus (SIV)-infected rhesus macaque. This analysis revealed rLCV orthologues of the latency-associated EBV circular RNAs circRPMS1_E4_E3a and circEBNA_U. Also identified in two samples displaying unusually high lytic gene expression was a novel rLCV circRNA that contains both conserved and rLCV-specific RPMS1 exons and whose backsplice junctions flank an rLCV lytic origin of replication (OriLyt). Analysis of a lytic infection model for the murid herpesvirus 68 (MHV68) rhadinovirus identified a cluster of circRNAs near an MHV68 lytic origin of replication, with the most abundant of these, circM11_ORF69, spanning the OriLyt. Lastly, analysis of KSHV latency and reactivation models revealed the latency associated circRNA originating from the vIRF4 gene as the predominant viral circRNA. Together, the results of this study broaden our appreciation for circRNA repertoires in the and genera of gammaherpesviruses and provide evolutionary support for viral circRNA functions in latency and viral replication. Infection with oncogenic gammaherpesviruses leads to long-term viral persistence through a dynamic interplay between the virus and the host immune system. Critical for remodeling of the host cell environment after the immune responses are viral noncoding RNAs that modulate host signaling pathways without attracting adaptive immune recognition. Despite the importance of noncoding RNAs in persistent infection, the circRNA class of noncoding RNAs has only recently been identified in gammaherpesviruses. Accordingly, their roles in virus infection and associated oncogenesis are unknown. Here we report evolutionary conservation of EBV-encoded circRNAs determined by assessing the circRNAome in rLCV-infected lymphomas from an SIV-infected rhesus macaque, and we report latent and lytic circRNAs from KSHV and MHV68. These experiments demonstrate utilization of the circular RNA class of RNAs across 4 members of the gammaherpesvirus subfamily, and they identify orthologues and potential homoplastic circRNAs, implying conserved circRNA functions in virus biology and associated malignancies.
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http://dx.doi.org/10.1128/JVI.01952-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401440PMC
March 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

The Epstein Barr virus circRNAome.

PLoS Pathog 2018 08 6;14(8):e1007206. Epub 2018 Aug 6.

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

Our appreciation for the extent of Epstein Barr virus (EBV) transcriptome complexity continues to grow through findings of EBV encoded microRNAs, new long non-coding RNAs as well as the more recent discovery of over a hundred new polyadenylated lytic transcripts. Here we report an additional layer to the EBV transcriptome through the identification of a repertoire of latent and lytic viral circular RNAs. Utilizing RNase R-sequencing with cell models representing latency types I, II, and III, we identified EBV encoded circular RNAs expressed from the latency Cp promoter involving backsplicing from the W1 and W2 exons to the C1 exon, from the EBNA BamHI U fragment exon, and from the latency long non-coding RPMS1 locus. In addition, we identified circular RNAs expressed during reactivation including backsplicing from exon 8 to exon 2 of the LMP2 gene and a highly expressed circular RNA derived from intra-exonic backsplicing within the BHLF1 gene. While expression of most of these circular RNAs was found to depend on the EBV transcriptional program utilized and the transcription levels of the associated loci, expression of LMP2 exon 8 to exon 2 circular RNA was found to be cell model specific. Altogether we identified over 30 unique EBV circRNAs candidates and we validated and determined the structural features, expression profiles and nuclear/cytoplasmic distributions of several predominant and notable viral circRNAs. Further, we show that two of the EBV circular RNAs derived from the RPMS1 locus are detected in EBV positive clinical stomach cancer specimens. This study increases the known EBV latency and lytic transcriptome repertoires to include viral circular RNAs and it provides an essential foundation and resource for investigations into the functions and roles of this new class of EBV transcripts in EBV biology and diseases.
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http://dx.doi.org/10.1371/journal.ppat.1007206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095625PMC
August 2018

Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice.

PLoS Pathog 2018 02 1;14(2):e1006843. Epub 2018 Feb 1.

Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, United States of America.

Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host.
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http://dx.doi.org/10.1371/journal.ppat.1006843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811070PMC
February 2018

Crystal Structure of Cleaved Serp-1, a Myxomavirus-Derived Immune Modulating Serpin: Structural Design of Serpin Reactive Center Loop Peptides with Improved Therapeutic Function.

Biochemistry 2018 02 6;57(7):1096-1107. Epub 2018 Feb 6.

Department of Medicine, Divisions of Cardiovascular Medicine and Rheumatology, University of Florida , Gainesville, Florida 32610-0277, United States.

The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculitis, lupus, viral sepsis, and transplant. Serp-1 has also been tested successfully in a Phase IIa clinical trial in unstable angina, representing a "first-in-class" therapeutic. Recently, peptides derived from the reactive center loop (RCL) have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-1 and the RCL peptides lose activity in MHV68-infected mice after antibiotic suppression of intestinal microbiota. Here, we utilize a structure-guided approach to design and test a series of next-generation RCL peptides with improved therapeutic potential that is not reduced when the peptides are combined with antibiotic treatments. The crystal structure of cleaved Serp-1 was determined to 2.5 Å resolution and reveals a classical serpin structure with potential for serpin-derived RCL peptides to bind and inhibit mammalian serpins, plasminogen activator inhibitor 1 (PAI-1), anti-thrombin III (ATIII), and α-1 antitrypsin (A1AT), and target proteases. Using in silico modeling of the Serp-1 RCL peptide, S-7, we designed several modified RCL peptides that were predicted to have stronger interactions with human serpins because of the larger number of stabilizing hydrogen bonds. Two of these peptides (MPS7-8 and -9) displayed extended activity, improving survival where activity was previously lost in antibiotic-treated MHV68-infected mice (P < 0.0001). Mass spectrometry and kinetic assays suggest interaction of the peptides with ATIII, A1AT, and target proteases in mouse and human plasma. In summary, we present the next step toward the development of a promising new class of anti-inflammatory serpin-based therapeutics.
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http://dx.doi.org/10.1021/acs.biochem.7b01171DOI Listing
February 2018

IκB Kinase ε Is an NFATc1 Kinase that Inhibits T Cell Immune Response.

Cell Rep 2016 07 23;16(2):405-418. Epub 2016 Jun 23.

Department of Molecular Microbiology and Immunology, Norris Comprehensive Cancer Center, University of Southern California, 1441 Eastlake Avenue, Los Angeles, CA 90033, USA. Electronic address:

Activation of nuclear factor of activated T cells (NFAT) is crucial for immune responses. IKKε is an IκB kinase (IKK)-related kinase, and the function of IKKε remains obscure in T cells, despite its abundant expression. We report that IKKε inhibits NFAT activation and T cell responses by promoting NFATc1 phosphorylation. During T cell activation, IKKε was transiently activated to phosphorylate NFATc1. Loss of IKKε elevated T cell antitumor and antiviral immunity and, therefore, reduced tumor development and persistent viral infection. IKKε was activated in CD8(+) T cells of mice bearing melanoma or persistently infected with a model herpesvirus. These results collectively show that IKKε promotes NFATc1 phosphorylation and inhibits T cell responses, identifying IKKε as a crucial negative regulator of T cell activation and a potential target for immunotherapy.
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http://dx.doi.org/10.1016/j.celrep.2016.05.083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293007PMC
July 2016

Recent advances in understanding norovirus pathogenesis.

J Med Virol 2016 11 5;88(11):1837-43. Epub 2016 May 5.

Department of Molecular Genetics and Microbiology, College of Medicine, Emerging Pathogens Institute, University of Florida, Gainesville, Florida.

Noroviruses constitute a family of ubiquitous and highly efficient human pathogens. In spite of decades of dedicated research, human noroviruses remain a major cause of gastroenteritis and severe diarrheal disease around the world. Recent findings have begun to unravel the complex mechanisms that regulate norovirus pathogenesis and persistent infection, including the important interplay between the virus, the host immune system, and commensal bacteria. Herein, we will summarize recent research developments regarding norovirus cell tropism, the use of M cells, and commensal bacteria to facilitate norovirus infection, and virus, host, and bacterial determinants of persistent norovirus infections. J. Med. Virol. 88:1837-1843, 2016. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jmv.24559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5203933PMC
November 2016

A Gammaherpesvirus Noncoding RNA Is Essential for Hematogenous Dissemination and Establishment of Peripheral Latency.

mSphere 2016 Apr 2;1(2). Epub 2016 Mar 2.

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

Recent intense investigations have uncovered important functions for a diverse array of novel noncoding RNA (ncRNA) species, including microRNAs (miRNAs) and long noncoding RNAs. Not surprisingly, viruses from multiple families have evolved to encode their own regulatory RNAs; however, the specific functions of these ncRNAs are largely unknown. The human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are highly ubiquitous pathogens that are associated with the development of a wide range of malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma, and Kaposi's sarcoma. Like EBV and KSHV, murine gammaherpesvirus 68 (MHV68) establishes lifelong latency in B cells and is associated with lymphoproliferative disease and lymphoma. Similar to the EBV-encoded small RNA (EBER)-1 and -2, MHV68 encodes eight 200- to 250-nucleotide polymerase III-transcribed ncRNAs called TMERs (tRNA-miRNA-encoded RNAs), which are highly expressed in latently infected cells and lymphoproliferative disease. To define the contribution of TMERs to MHV68 biology, we generated a panel of individual TMER mutant viruses. Through comprehensive analyses, we identified TMER4 as a key mediator of virus dissemination. The TMER4 mutant virus replicated normally in lungs and spread with normal kinetics and distribution to lung-draining lymph nodes, but it was significantly attenuated for infection of circulating blood cells and for latency establishment at peripheral sites. Notably, TMER4 stem-loops but not miRNAs were essential for wild-type TMER4 activity. Thus, these findings revealed a crucial miRNA-independent function of the TMER4 ncRNA in MHV68 hematogenous dissemination and latency establishment.

Importance: Noncoding RNAs (ncRNAs) represent an intriguing and diverse class of molecules that are now recognized for their participation in a wide array of cellular processes. Viruses from multiple families have evolved to encode their own such regulatory RNAs; however, the specific functions of these ncRNAs are largely unknown. Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are ubiquitous human pathogens that are associated with the development of numerous malignancies. Like EBV and KSHV, murine gammaherpesvirus 68 (MHV68) establishes lifelong latency in B cells and is associated with lymphomagenesis. The work described here reveals that the MHV68 ncRNA TMER4 acts at a critical bottleneck in local lymph nodes to facilitate hematogenous dissemination of the virus and establishment of latency at peripheral sites.
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http://dx.doi.org/10.1128/mSphere.00105-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838037PMC
April 2016

A Gammaherpesvirus Noncoding RNA Is Essential for Hematogenous Dissemination and Establishment of Peripheral Latency.

mSphere 2016 Apr 2;1(2). Epub 2016 Mar 2.

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

Recent intense investigations have uncovered important functions for a diverse array of novel noncoding RNA (ncRNA) species, including microRNAs (miRNAs) and long noncoding RNAs. Not surprisingly, viruses from multiple families have evolved to encode their own regulatory RNAs; however, the specific functions of these ncRNAs are largely unknown. The human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are highly ubiquitous pathogens that are associated with the development of a wide range of malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma, and Kaposi's sarcoma. Like EBV and KSHV, murine gammaherpesvirus 68 (MHV68) establishes lifelong latency in B cells and is associated with lymphoproliferative disease and lymphoma. Similar to the EBV-encoded small RNA (EBER)-1 and -2, MHV68 encodes eight 200- to 250-nucleotide polymerase III-transcribed ncRNAs called TMERs (tRNA-miRNA-encoded RNAs), which are highly expressed in latently infected cells and lymphoproliferative disease. To define the contribution of TMERs to MHV68 biology, we generated a panel of individual TMER mutant viruses. Through comprehensive analyses, we identified TMER4 as a key mediator of virus dissemination. The TMER4 mutant virus replicated normally in lungs and spread with normal kinetics and distribution to lung-draining lymph nodes, but it was significantly attenuated for infection of circulating blood cells and for latency establishment at peripheral sites. Notably, TMER4 stem-loops but not miRNAs were essential for wild-type TMER4 activity. Thus, these findings revealed a crucial miRNA-independent function of the TMER4 ncRNA in MHV68 hematogenous dissemination and latency establishment.

Importance: Noncoding RNAs (ncRNAs) represent an intriguing and diverse class of molecules that are now recognized for their participation in a wide array of cellular processes. Viruses from multiple families have evolved to encode their own such regulatory RNAs; however, the specific functions of these ncRNAs are largely unknown. Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are ubiquitous human pathogens that are associated with the development of numerous malignancies. Like EBV and KSHV, murine gammaherpesvirus 68 (MHV68) establishes lifelong latency in B cells and is associated with lymphomagenesis. The work described here reveals that the MHV68 ncRNA TMER4 acts at a critical bottleneck in local lymph nodes to facilitate hematogenous dissemination of the virus and establishment of latency at peripheral sites.
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http://dx.doi.org/10.1128/mSphere.00105-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838037PMC
April 2016

Human norovirus culture in B cells.

Nat Protoc 2015 Dec 29;10(12):1939-47. Epub 2015 Oct 29.

Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA.

Human noroviruses (HuNoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HuNoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HuNoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-Sydney HuNoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HuNoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. Analysis of infection or attachment samples, including RNA extraction and RT-qPCR, requires ∼6 h.
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http://dx.doi.org/10.1038/nprot.2015.121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4689599PMC
December 2015

Emerging Roles of Herpesvirus microRNAs During In Vivo Infection and Pathogenesis.

Curr Pathobiol Rep 2015;3(3):209-217

Department of Molecular Genetics & Microbiology, College of Medicine, UF Health Cancer Center, University of Florida, 1200 Newell Dr., R2-291, Gainesville, FL 32610 USA.

Herpesviridae constitutes a large family of double-stranded DNA viruses that are associated with a wide range of diseases, including herpetic lesions, birth defects, and cancer. Herpesviruses establish lifelong latent infections in part because they are exceptionally adept at modulating the virus/host interface. New insights into the numerous roles of microRNAs (miRNAs) in cell biology, along with the recent appreciation that nearly every host transcript is targeted by at least one miRNA, has fundamentally changed our conceptualization of the virus/host relationship. The identification of miRNAs expressed from nearly all human herpesvirus genomes has led to the speculation that these short non-coding transcripts play essential roles in herpesvirus biology. Because the activity of miRNAs depends upon the transcriptome of the cell in which they are expressed, in vivo systems will be essential for defining the true biological relevance of herpesvirus miRNAs. This review will specifically focus on experimental systems which have investigated the functional role of herpesvirus-encoded miRNAs in viral biology and pathogenesis in vivo.
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http://dx.doi.org/10.1007/s40139-015-0085-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522463PMC
January 2015

Recombinant Murine Gamma Herpesvirus 68 Carrying KSHV G Protein-Coupled Receptor Induces Angiogenic Lesions in Mice.

PLoS Pathog 2015 Jun 24;11(6):e1005001. Epub 2015 Jun 24.

Department of Molecular Microbiology and Immunology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.

Human gamma herpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are capable of inducing tumors, particularly in in immune-compromised individuals. Due to the stringent host tropism, rodents are resistant to infection by human gamma herpesviruses, creating a significant barrier for the in vivo study of viral genes that contribute to tumorigenesis. The closely-related murine gamma herpesvirus 68 (γHV68) efficiently infects laboratory mouse strains and establishes robust persistent infection without causing apparent disease. Here, we report that a recombinant γHV68 carrying the KSHV G protein-coupled receptor (kGPCR) in place of its murine counterpart induces angiogenic tumors in infected mice. Although viral GPCRs are conserved in all gamma herpesviruses, kGPCR potently activated downstream signaling and induced tumor formation in nude mouse, whereas γHV68 GPCR failed to do so. Recombinant γHV68 carrying kGPCR demonstrated more robust lytic replication ex vivo than wild-type γHV68, although both viruses underwent similar acute and latent infection in vivo. Infection of immunosuppressed mice with γHV68 carrying kGPCR, but not wild-type γHV68, induced tumors in mice that exhibited angiogenic and inflammatory features shared with human Kaposi's sarcoma. Immunohistochemistry staining identified abundant latently-infected cells and a small number of cells supporting lytic replication in tumor tissue. Thus, mouse infection with a recombinant γHV68 carrying kGPCR provides a useful small animal model for tumorigenesis induced by a human gamma herpesvirus gene in the setting of a natural course of infection.
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http://dx.doi.org/10.1371/journal.ppat.1005001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4479558PMC
June 2015

Gammaherpesvirus small noncoding RNAs are bifunctional elements that regulate infection and contribute to virulence in vivo.

mBio 2015 Feb 17;6(1):e01670-14. Epub 2015 Feb 17.

Department of Immunology & Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA

Unlabelled: Many viruses express noncoding RNAs (ncRNAs). The gammaherpesviruses (γHVs), including Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and murine γHV68, each contain multiple ncRNA genes, including microRNAs (miRNAs). While these ncRNAs can regulate multiple host and viral processes in vitro, the genetic contribution of these RNAs to infection and pathogenesis remains largely unknown. To study the functional contribution of these RNAs to γHV infection, we have used γHV68, a small-animal model of γHV pathogenesis. γHV68 encodes eight small hybrid ncRNAs that contain both tRNA-like elements and functional miRNAs. These genes are transcribed by RNA polymerase III and are referred to as the γHV68 TMERs (tRNA-miRNA-encoded RNAs). To determine the total concerted genetic contribution of these ncRNAs to γHV acute infection and pathogenesis, we generated and characterized a recombinant γHV68 strain devoid of all eight TMERs. TMER-deficient γHV68 has wild-type levels of lytic replication in vitro and normal establishment of latency in B cells early following acute infection in vivo. In contrast, during acute infection of immunodeficient mice, TMER-deficient γHV68 has reduced virulence in a model of viral pneumonia, despite having an enhanced frequency of virus-infected cells. Strikingly, expression of a single viral tRNA-like molecule, in the absence of all other virus-encoded TMERs and miRNAs, reverses both attenuation in virulence and enhanced frequency of infected cells. These data show that γHV ncRNAs play critical roles in acute infection and virulence in immunocompromised hosts and identify these RNAs as a new potential target to modulate γHV-induced infection and pathogenesis.

Importance: The gammaherpesviruses (γHVs) are a subfamily of viruses associated with chronic inflammatory diseases and cancer, particularly in immunocompromised individuals. These viruses uniformly encode multiple types of noncoding RNAs (ncRNAs) that are not translated into proteins. It remains unclear how virus-expressed ncRNAs influence the course and outcome of infection in vivo. Here, we generated a mouse γHV that lacks the expression of multiple ncRNAs. Notably, this mutant virus is critically impaired in the ability to cause disease in immunocompromised hosts yet shows a paradoxical increase in infected cells early during infection in these hosts. While the original mouse virus encodes multiple ncRNAs, the expression of a single domain of one ncRNA can partially reverse the defects of the mutant virus. These studies demonstrate that γHV ncRNAs can directly contribute to virus-induced disease in vivo and that these RNAs may be multifunctional, allowing the opportunity to specifically interfere with different functional domains of these RNAs.
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http://dx.doi.org/10.1128/mBio.01670-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4337559PMC
February 2015

Enteric bacteria promote human and mouse norovirus infection of B cells.

Science 2014 Nov;346(6210):755-9

Department of Molecular Genetics and Microbiology, Emerging Pathogens Institute, College of Medicine, University of Florida, Gainesville, FL, USA.

The cell tropism of human noroviruses and the development of an in vitro infection model remain elusive. Although susceptibility to individual human norovirus strains correlates with an individual's histo-blood group antigen (HBGA) profile, the biological basis of this restriction is unknown. We demonstrate that human and mouse noroviruses infected B cells in vitro and likely in vivo. Human norovirus infection of B cells required the presence of HBGA-expressing enteric bacteria. Furthermore, mouse norovirus replication was reduced in vivo when the intestinal microbiota was depleted by means of oral antibiotic administration. Thus, we have identified B cells as a cellular target of noroviruses and enteric bacteria as a stimulatory factor for norovirus infection, leading to the development of an in vitro infection model for human noroviruses.
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http://dx.doi.org/10.1126/science.1257147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4401463PMC
November 2014

Virus-encoded microRNAs facilitate gammaherpesvirus latency and pathogenesis in vivo.

mBio 2014 May 27;5(3):e00981-14. Epub 2014 May 27.

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

Unlabelled: Gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV, or HHV-8), and murine gammaherpesvirus 68 (MHV68, γHV68, or MuHV-4), are B cell-tropic pathogens that each encode at least 12 microRNAs (miRNAs). It is predicted that these regulatory RNAs facilitate infection by suppressing host target genes involved in a wide range of key cellular pathways. However, the precise contribution that gammaherpesvirus miRNAs make to viral life cycle and pathogenesis in vivo is unknown. MHV68 infection of mice provides a highly useful system to dissect the function of specific viral elements in the context of both asymptomatic infection and disease. Here, we report (i) analysis of in vitro and in vivo MHV68 miRNA expression, (ii) generation of an MHV68 miRNA mutant with reduced expression of all 14 pre-miRNA stem-loops, and (iii) comprehensive phenotypic characterization of the miRNA mutant virus in vivo. The profile of MHV68 miRNAs detected in infected cell lines varied with cell type and did not fully recapitulate the profile from cells latently infected in vivo. The miRNA mutant virus, MHV68.Zt6, underwent normal lytic replication in vitro and in vivo, demonstrating that the MHV68 miRNAs are dispensable for acute replication. During chronic infection, MHV68.Zt6 was attenuated for latency establishment, including a specific defect in memory B cells. Finally, MHV68.Zt6 displayed a striking attenuation in the development of lethal pneumonia in mice deficient in IFN-γ. These data indicate that the MHV68 miRNAs may facilitate virus-driven maturation of infected B cells and implicate the miRNAs as a critical determinant of gammaherpesvirus-associated disease.

Importance: Gammaherpesviruses such as EBV and KSHV are widespread pathogens that establish lifelong infections and are associated with the development of numerous types of diseases, including cancer. Gammaherpesviruses encode many small noncoding RNAs called microRNAs (miRNAs). It is predicted that gammaherpesvirus miRNAs facilitate infection and disease by suppressing host target transcripts involved in a wide range of key cellular pathways; however, the precise contribution that these regulatory RNAs make to in vivo virus infection and pathogenesis is unknown. Here, we generated a mutated form of murine gammaherpesvirus (MHV68) to dissect the function of gammaherpesvirus miRNAs in vivo. We demonstrate that the MHV68 miRNAs were dispensable for short-term virus replication but were important for establishment of lifelong infection in the key virus reservoir of memory B cells. Moreover, the MHV68 miRNAs were essential for the development of virus-associated pneumonia, implicating them as a critical component of gammaherpesvirus-associated disease.
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http://dx.doi.org/10.1128/mBio.00981-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4045068PMC
May 2014

A gammaherpesvirus Bcl-2 ortholog blocks B cell receptor-mediated apoptosis and promotes the survival of developing B cells in vivo.

PLoS Pathog 2014 Feb 6;10(2):e1003916. Epub 2014 Feb 6.

Department of Molecular Genetics & Microbiology and UF Shands Cancer Center, College of Medicine, University of Florida, Gainesville, Florida, United States of America.

Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) establish lifelong latency in their hosts and are associated with the development of several types of malignancies, including a subset of B cell lymphomas. These viruses are thought to co-opt the process of B cell differentiation to latently infect a fraction of circulating memory B cells, resulting in the establishment of a stable latency setpoint. However, little is known about how this infected memory B cell compartment is maintained throughout the life of the host. We have previously demonstrated that immature and transitional B cells are long-term latency reservoirs for murine gammaherpesvirus 68 (MHV68), suggesting that infection of developing B cells contributes to the maintenance of lifelong latency. During hematopoiesis, immature and transitional B cells are subject to B cell receptor (BCR)-mediated negative selection, which results in the clonal deletion of autoreactive B cells. Interestingly, numerous gammaherpesviruses encode homologs of the anti-apoptotic protein Bcl-2, suggesting that virus inhibition of apoptosis could subvert clonal deletion. To test this, we quantified latency establishment in mice inoculated with MHV68 vBcl-2 mutants. vBcl-2 mutant viruses displayed a marked decrease in the frequency of immature and transitional B cells harboring viral genome, but this attenuation could be rescued by increased host Bcl-2 expression. Conversely, vBcl-2 mutant virus latency in early B cells and mature B cells, which are not targets of negative selection, was remarkably similar to wild-type virus. Finally, in vivo depletion of developing B cells during chronic infection resulted in decreased mature B cell latency, demonstrating a key role for developing B cells in the maintenance of lifelong latency. Collectively, these findings support a model in which gammaherpesvirus latency in circulating mature B cells is sustained in part through the recurrent infection and vBcl-2-mediated survival of developing B cells.
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http://dx.doi.org/10.1371/journal.ppat.1003916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3916410PMC
February 2014

Myxomavirus-derived serpin prolongs survival and reduces inflammation and hemorrhage in an unrelated lethal mouse viral infection.

Antimicrob Agents Chemother 2013 Sep 17;57(9):4114-27. Epub 2013 Jun 17.

Divisions of Cardiology and Rheumatology, Department of Medicine, University of Florida, Gainesville, Florida, USA.

Lethal viral infections produce widespread inflammation with vascular leak, clotting, and bleeding (disseminated intravascular coagulation [DIC]), organ failure, and high mortality. Serine proteases in clot-forming (thrombotic) and clot-dissolving (thrombolytic) cascades are activated by an inflammatory cytokine storm and also can induce systemic inflammation with loss of normal serine protease inhibitor (serpin) regulation. Myxomavirus secretes a potent anti-inflammatory serpin, Serp-1, that inhibits clotting factor X (fX) and thrombolytic tissue- and urokinase-type plasminogen activators (tPA and uPA) with anti-inflammatory activity in multiple animal models. Purified serpin significantly improved survival in a murine gammaherpesvirus 68 (MHV68) infection in gamma interferon receptor (IFN-γR) knockout mice, a model for lethal inflammatory vasculitis. Treatment of MHV68-infected mice with neuroserpin, a mammalian serpin that inhibits only tPA and uPA, was ineffective. Serp-1 reduced virus load, lung hemorrhage, and aortic, lung, and colon inflammation in MHV68-infected mice and also reduced virus load. Neuroserpin suppressed a wide range of immune spleen cell responses after MHV68 infection, while Serp-1 selectively increased CD11c(+) splenocytes (macrophage and dendritic cells) and reduced CD11b(+) tissue macrophages. Serp-1 altered gene expression for coagulation and inflammatory responses, whereas neuroserpin did not. Serp-1 treatment was assessed in a second viral infection, mouse-adapted Zaire ebolavirus in wild-type BALB/c mice, with improved survival and reduced tissue necrosis. In summary, treatment with this unique myxomavirus-derived serpin suppresses systemic serine protease and innate immune responses caused by unrelated lethal viral infections (both RNA and DNA viruses), providing a potential new therapeutic approach for treatment of lethal viral sepsis.
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http://dx.doi.org/10.1128/AAC.02594-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754305PMC
September 2013

Unbiased mutagenesis of MHV68 LANA reveals a DNA-binding domain required for LANA function in vitro and in vivo.

PLoS Pathog 2012 Sep 6;8(9):e1002906. Epub 2012 Sep 6.

Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, United States of America.

The Latency-Associated Nuclear Antigen (LANA), encoded by ORF73, is a conserved gene among the γ2-herpesviruses (rhadinoviruses). The Kaposi's Sarcoma-Associated Herpesvirus (KSHV) LANA is consistently expressed in KSHV-associated malignancies. In the case of the rodent γ2-herpesvirus, murine gammaherpesvirus 68 (MHV68), the LANA homolog (mLANA) is required for efficient virus replication, reactivation from latency and immortalization of murine fetal liver-derived B cells. To gain insights into mLANA function(s), knowing that KSHV LANA binds DNA and can modulate transcription of a variety of promoters, we sought out and identified a mLANA-responsive promoter which maps to the terminal repeat (TR) of MHV68. Notably, mLANA strongly repressed activity from this promoter. We extended these analyses to demonstrate direct, sequence-specific binding of recombinant mLANA to TR DNA by DNase I footprinting. To assess whether the DNA-binding and/or transcription modulating function is important in the known mLANA phenotypes, we generated an unbiased library of mLANA point mutants using error-prone PCR, and screened a large panel of mutants for repression of the mLANA-responsive promoter to identify loss of function mutants. Notably, among the mutant mLANA proteins recovered, many of the mutations are in a predicted EBNA-1-like DNA-binding domain. Consistent with this prediction, those tested displayed loss of DNA binding activity. We engineered six of these mLANA mutants into the MHV68 genome and tested the resulting mutant viruses for: (i) replication fitness; (ii) efficiency of latency establishment; and (iii) reactivation from latency. Interestingly, each of these mLANA-mutant viruses exhibited phenotypes similar to the mLANA-null mutant virus, indicating that DNA-binding is critical for mLANA function.
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http://dx.doi.org/10.1371/journal.ppat.1002906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3435236PMC
September 2012

Immature and transitional B cells are latency reservoirs for a gammaherpesvirus.

J Virol 2010 Dec 6;84(24):13045-52. Epub 2010 Oct 6.

Center for Molecular and Tumor Virology, Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, USA.

Gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 [HHV-8]), Epstein-Barr virus (EBV), and murine gammaherpesvirus 68 (MHV68; also known as gammaherpesvirus 68 [γHV68] or murine herpesvirus 4 [MuHV-4]), establish lifelong latency in the resting memory B cell compartment. However, little is known about how this reservoir of infected mature B cells is maintained for the life of the host. In the context of a normal immune system, the mature B cell pool is naturally maintained by the renewable populations of developing B cells that arise from hematopoiesis. Thus, recurrent infection of these developing B cell populations could allow the virus continual access to the B cell lineage and, subsequent to differentiation, the memory B cell compartment. To begin to address this hypothesis, we examined whether MHV68 establishes latency in developing B cells during a normal course of infection. In work described here, we demonstrate the presence of viral genome in bone marrow pro-pre-B cells and immature B cells during early latency and immature B cells during long-term latency. Further, we show that transitional B cells in the spleen are latently infected and express the latency-associated nuclear antigen (LANA) throughout chronic infection. Because developing B cells normally exhibit a short life span and a high rate of turnover, these findings suggest a model in which gammaherpesviruses may gain access to the mature B cell compartment by recurrent seeding of developing B cells.
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http://dx.doi.org/10.1128/JVI.01455-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3004345PMC
December 2010

Murine gamma-herpesvirus 68 hijacks MAVS and IKKbeta to initiate lytic replication.

PLoS Pathog 2010 Jul 29;6(7):e1001001. Epub 2010 Jul 29.

Department of Microbiology, UT Southwestern Medical Center, Dallas, Texas, USA.

Upon viral infection, the mitochondrial antiviral signaling (MAVS)-IKKbeta pathway is activated to restrict viral replication. Manipulation of immune signaling events by pathogens has been an outstanding theme of host-pathogen interaction. Here we report that the loss of MAVS or IKKbeta impaired the lytic replication of gamma-herpesvirus 68 (gammaHV68), a model herpesvirus for human Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. gammaHV68 infection activated IKKbeta in a MAVS-dependent manner; however, IKKbeta phosphorylated and promoted the transcriptional activation of the gammaHV68 replication and transcription activator (RTA). Mutational analyses identified IKKbeta phosphorylation sites, through which RTA-mediated transcription was increased by IKKbeta, within the transactivation domain of RTA. Moreover, the lytic replication of recombinant gammaHV68 carrying mutations within the IKKbeta phosphorylation sites was greatly impaired. These findings support the conclusion that gammaHV68 hijacks the antiviral MAVS-IKKbeta pathway to promote viral transcription and lytic infection, representing an example whereby viral replication is coupled to host immune activation.
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http://dx.doi.org/10.1371/journal.ppat.1001001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2912392PMC
July 2010