RESUMO
Kaposi's sarcoma-associated herpesvirus is the etiologic agent of Kaposi's sarcoma and two B-cell malignancies. Recent advancements in sequencing technologies have led to high resolution transcriptomes for several human herpesviruses that densely encode genes on both strands. However, for KSHV progress remained limited due to the overall low percentage of KSHV transcripts, even during lytic replication. To address this challenge, we have developed a target enrichment method to increase the KSHV-specific reads for both short- and long-read sequencing platforms. Furthermore, we combined this approach with the Transcriptome Resolution through Integration of Multi-platform Data (TRIMD) pipeline developed previously to annotate transcript structures. TRIMD first builds a scaffold based on long-read sequencing and validates each transcript feature with supporting evidence from Illumina RNA-Seq and deepCAGE sequencing data. Our stringent innovative approach identified 994 unique KSHV transcripts, thus providing the first high-density KSHV lytic transcriptome. We describe a plethora of novel coding and non-coding KSHV transcript isoforms with alternative untranslated regions, splice junctions and open-reading frames, thus providing deeper insights on gene expression regulation of KSHV. Interestingly, as described for Epstein-Barr virus, we identified transcription start sites that augment long-range transcription and may increase the number of latency-associated genes potentially expressed in KS tumors.
Assuntos
Processamento Alternativo , Herpesvirus Humano 8 , Transcriptoma , Herpesvirus Humano 8/genética , Humanos , Transcriptoma/genética , Transcrição Gênica , Regulação Viral da Expressão Gênica , Fases de Leitura Aberta/genética , Sequenciamento de Nucleotídeos em Larga Escala , Sarcoma de Kaposi/virologia , Sarcoma de Kaposi/genética , RNA Viral/genética , RNA Viral/metabolismoRESUMO
The germinal center (GC) plays a central role in the generation of antigen-specific B cells and antibodies. Tight regulation of the GC is essential due to the inherent risks of tumorigenesis and autoimmunity posed by inappropriate GC B cell processes. Gammaherpesviruses such as Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) utilize numerous armaments to drive infected naïve B cells, independent of antigen, through GC reactions to expand the latently infected B cell population and establish a stable latency reservoir. We previously demonstrated that the MHV68 microRNA (miRNA) mghv-miR-M1-7-5p represses host EWSR1 (Ewing sarcoma breakpoint region 1) to promote B cell infection. EWSR1 is a transcription and splicing regulator that is recognized for its involvement as a fusion protein in Ewing sarcoma. A function for EWSR1 in B cell responses has not been previously reported. Here, we demonstrate that 1) B cell-specific deletion of EWSR1 had no effect on generation of mature B cell subsets or basal immunoglobulin levels in naïve mice, 2) repression or ablation of EWSR1 in B cells promoted expansion of MHV68 latently infected GC B cells, and 3) B cell-specific deletion of EWSR1 during a normal immune response to nonviral antigen resulted in significantly elevated numbers of antigen-specific GC B cells, plasma cells, and circulating antibodies. Notably, EWSR1 deficiency did not affect the proliferation or survival of GC B cells but instead resulted in the generation of increased numbers of precursor GC B cells. Cumulatively, these findings demonstrate that EWSR1 is a negative regulator of B cell responses.
Assuntos
Linfócitos B , Gammaherpesvirinae , Centro Germinativo , Infecções por Herpesviridae , MicroRNAs , Proteína EWS de Ligação a RNA , Infecções Tumorais por Vírus , Animais , Linfócitos B/imunologia , Linfócitos B/virologia , Gammaherpesvirinae/genética , Gammaherpesvirinae/fisiologia , Deleção de Genes , Centro Germinativo/imunologia , Centro Germinativo/virologia , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/virologia , Latência ViralRESUMO
IMPORTANCE: The human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are etiologic agents of numerous B cell lymphomas. A hallmark of gammaherpesvirus infection is their ability to establish lifelong latency in B cells. However, the specific mechanisms that mediate chronic infection in B cells in vivo remain elusive. Cellular E3 ubiquitin ligases regulate numerous biological processes by catalyzing ubiquitylation and modifying protein location, function, or half-life. Many viruses hijack host ubiquitin ligases to evade antiviral host defense and promote viral fitness. Here, we used the murine gammaherpesvirus 68 in vivo system to demonstrate that the E3 ligase Cul4b is essential for this virus to establish latency in germinal center B cells. These findings highlight an essential role for this E3 ligase in promoting chronic gammaherpesvirus infection in vivo and suggest that targeted inhibition of E3 ligases may provide a novel and effective intervention strategy against gammaherpesvirus-associated diseases.
Assuntos
Linfócitos B , Gammaherpesvirinae , Infecções por Herpesviridae , Infecção Persistente , Animais , Camundongos , Linfócitos B/enzimologia , Linfócitos B/metabolismo , Linfócitos B/virologia , Proteínas Culina/metabolismo , Gammaherpesvirinae/fisiologia , Centro Germinativo/citologia , Centro Germinativo/virologia , Infecções por Herpesviridae/enzimologia , Infecções por Herpesviridae/virologia , Infecção Persistente/enzimologia , Infecção Persistente/virologia , Ubiquitinas/metabolismo , Latência ViralRESUMO
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.
Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/imunologia , MicroRNAs/genética , RNA Viral/genética , Complexo de Inativação Induzido por RNA/metabolismo , Transcriptoma , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/patologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/genética , Humanos , Complexo de Inativação Induzido por RNA/genéticaRESUMO
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.
Assuntos
Gammaherpesvirinae/genética , RNA não Traduzido , RNA Viral , Liberação de Vírus/genética , Animais , Células Cultivadas , Infecções por Herpesviridae/virologia , Camundongos , Conformação de Ácido Nucleico , RNA não Traduzido/química , RNA não Traduzido/genética , RNA não Traduzido/fisiologia , RNA Viral/química , RNA Viral/genética , RNA Viral/fisiologia , Baço/citologia , Baço/virologia , Latência Viral/genéticaRESUMO
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.
Assuntos
Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/metabolismo , MicroRNAs/genética , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Animais , Regulação da Expressão Gênica , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Camundongos , RNA Mensageiro/genética , RNA Viral/genética , RNA Viral/metabolismo , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/genética , Replicação ViralRESUMO
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 Lymphocryptovirus and Rhadinovirus genera of gammaherpesviruses and provide evolutionary support for viral circRNA functions in latency and viral replication.IMPORTANCE 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.
Assuntos
Gammaherpesvirinae/genética , RNA/genética , Animais , Linhagem Celular , Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 8/genética , Humanos , Lymphocryptovirus/genética , Macaca mulatta , Masculino , RNA Circular , RNA Viral/genética , Rhadinovirus/genética , Vírus da Imunodeficiência Símia/genética , Latência Viral/genética , Replicação Viral/genéticaRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1006843.].
RESUMO
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.
Assuntos
Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 4/genética , RNA Viral/genética , RNA/genética , Latência Viral/genética , Linhagem Celular , Infecções por Vírus Epstein-Barr/genética , Humanos , RNA Circular , RNA não Traduzido/genéticaRESUMO
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.
Assuntos
Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/virologia , Pulmão/virologia , Macrófagos/virologia , Fases de Leitura Aberta , Baço/virologia , Proteínas Virais/metabolismo , Animais , Células da Medula Óssea/citologia , Células Cultivadas , Códon sem Sentido , DNA Recombinante/metabolismo , DNA Viral/metabolismo , Embrião de Mamíferos/citologia , Gammaherpesvirinae/crescimento & desenvolvimento , Gammaherpesvirinae/patogenicidade , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/patologia , Pulmão/imunologia , Pulmão/patologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Filogenia , Baço/imunologia , Baço/patologia , Carga Viral , Proteínas Virais/genética , Latência Viral , Replicação ViralRESUMO
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.
Assuntos
Fatores Imunológicos/química , Myxoma virus/química , Peptídeos/química , Serpinas/química , Proteínas Virais/química , Animais , Células CHO , Cricetulus , Cristalografia por Raios X , Humanos , Fatores Imunológicos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Peptídeos/farmacologia , Infecções por Poxviridae/virologia , Conformação Proteica , Coelhos , Serpinas/farmacologia , Proteínas Virais/farmacologiaRESUMO
Serpins regulate coagulation and inflammation, binding serine proteases in suicide-inhibitory complexes. Target proteases cleave the serpin reactive center loop scissile P1-P1' bond, resulting in serpin-protease suicide-inhibitory complexes. This inhibition requires a near full-length serpin sequence. Myxomavirus Serp-1 inhibits thrombolytic and thrombotic proteases, whereas mammalian neuroserpin (NSP) inhibits only thrombolytic proteases. Both serpins markedly reduce arterial inflammation and plaque in rodent models after single dose infusion. In contrast, Serp-1 but not NSP improves survival in a lethal murine gammaherpesvirus68 (MHV68) infection in interferon γ-receptor-deficient mice (IFNγR(-/-)). Serp-1 has also been successfully tested in a Phase 2a clinical trial. We postulated that proteolytic cleavage of the reactive center loop produces active peptide derivatives with expanded function. Eight peptides encompassing predicted protease cleavage sites for Serp-1 and NSP were synthesized and tested for inhibitory function in vitro and in vivo. In engrafted aorta, selected peptides containing Arg or Arg-Asn, not Arg-Met, with a 0 or +1 charge, significantly reduced plaque. Conversely, S-6 a hydrophobic peptide of NSP, lacking Arg or Arg-Asn with -4 charge, induced early thrombosis and mortality. S-1 and S-6 also significantly reduced CD11b(+) monocyte counts in mouse splenocytes. S-1 peptide had increased efficacy in plasminogen activator inhibitor-1 serpin-deficient transplants. Plaque reduction correlated with mononuclear cell activation. In a separate study, Serp-1 peptide S-7 improved survival in the MHV68 vasculitis model, whereas an inverse S-7 peptide was inactive. Reactive center peptides derived from Serp-1 and NSP with suitable charge and hydrophobicity have the potential to extend immunomodulatory functions of serpins.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Infecções por Herpesviridae/imunologia , Fatores Imunológicos , Proteínas de Membrana , Peptídeos , Rhadinovirus/imunologia , Vasculite/imunologia , Animais , Coagulação Sanguínea/imunologia , Modelos Animais de Doenças , Infecções por Herpesviridae/tratamento farmacológico , Humanos , Fatores Imunológicos/síntese química , Fatores Imunológicos/química , Fatores Imunológicos/imunologia , Células Jurkat , Proteínas de Membrana/síntese química , Proteínas de Membrana/química , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Knockout , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Vasculite/tratamento farmacológicoRESUMO
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.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/metabolismo , Rhadinovirus/genética , Infecções Tumorais por Vírus/virologia , Proteínas Virais/metabolismo , Latência Viral/fisiologia , Animais , Modelos Animais de Doenças , Proteínas de Ligação ao GTP/genética , Herpesvirus Humano 8/genética , Humanos , Camundongos , Neovascularização Patológica/virologia , Proteínas Virais/genética , Latência Viral/imunologiaRESUMO
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.
Assuntos
Linfócitos B/virologia , Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Infecções Tumorais por Vírus/imunologia , Latência Viral/imunologia , Animais , Apoptose/imunologia , Linfócitos B/citologia , Western Blotting , Diferenciação Celular/imunologia , Sobrevivência Celular/imunologia , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos B/imunologiaRESUMO
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.
Assuntos
Infecções por Caliciviridae/virologia , Enterócitos/virologia , Gastroenterite/virologia , Norovirus/patogenicidade , Animais , Antivirais/uso terapêutico , Infecções por Caliciviridae/tratamento farmacológico , Infecções por Caliciviridae/microbiologia , Técnicas de Cultura de Células , Interações Hospedeiro-Patógeno , Humanos , Intestinos/microbiologia , Intestinos/virologia , Camundongos , Norovirus/fisiologia , Simbiose , Tropismo Viral , Replicação ViralRESUMO
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.
Assuntos
Antígenos Virais/genética , Antígenos Virais/fisiologia , DNA/metabolismo , Gammaherpesvirinae/genética , Mutagênese/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Domínios e Motivos de Interação entre Proteínas/genética , Animais , Antígenos Virais/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação Viral da Expressão Gênica , Células HEK293 , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Humanos , Camundongos , Células NIH 3T3 , Proteínas Nucleares/química , Domínios e Motivos de Interação entre Proteínas/fisiologia , Repressão Psicológica , Sequências Repetidas Terminais/genéticaRESUMO
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.
Assuntos
Hemorragia/tratamento farmacológico , Doença pelo Vírus Ebola/tratamento farmacológico , Infecções por Herpesviridae/tratamento farmacológico , Infecções por Herpesviridae/mortalidade , Proteínas de Membrana/farmacologia , Myxoma virus/química , Animais , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Modelos Animais de Doenças , Ebolavirus , Fator X/antagonistas & inibidores , Fator X/metabolismo , Gammaherpesvirinae , Hemorragia/mortalidade , Hemorragia/patologia , Hemorragia/virologia , Doença pelo Vírus Ebola/mortalidade , Doença pelo Vírus Ebola/patologia , Doença pelo Vírus Ebola/virologia , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Inflamação/tratamento farmacológico , Inflamação/mortalidade , Inflamação/patologia , Inflamação/virologia , Interferon gama/deficiência , Interferon gama/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Proteínas de Membrana/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Myxoma virus/fisiologia , Neuropeptídeos/farmacologia , Serpinas/farmacologia , Análise de Sobrevida , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Vasculite/tratamento farmacológico , Vasculite/mortalidade , Vasculite/patologia , Vasculite/virologia , NeuroserpinaRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Quinase I-kappa B/metabolismo , Rhadinovirus/fisiologia , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Infecções por Herpesviridae , Quinase I-kappa B/imunologia , Camundongos , Fosforilação , Ativação Transcricional , Infecções Tumorais por VírusRESUMO
The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, γHV68, MuHV-4), are associated with numerous malignancies, including B cell lymphomas and nasopharyngeal carcinoma. These viruses employ numerous molecular strategies to colonize the host, including the expression of noncoding RNAs (ncRNAs). As the first viral ncRNAs identified, EBV-encoded RNA 1 and 2 (EBER1 and EBER2, respectively) have been investigated extensively for decades; however, their specific in vivo functions remain largely unknown. In work here, we used chimeric MHV68 viruses in an in vivo complementation system to test whether EBV EBER2 contributes to acute and/or chronic phases of infection. Expression of EBER2 derived from EBV strain B95-8 resulted in a significant expansion of latently infected B cells in vivo, which was accompanied by a decrease in virus-infected plasma cells. EBV strains typically carry one of two variants of EBER2, which differ primarily by a 5-nucleotide core polymorphism identified initially in the EBV strain M81. Strikingly, mutation of the 5 nucleotides that define this core polymorphism resulted in the loss of the infected B cell expansion and restored plasma cell infection. This work reveals that the B95-8 variant of EBER2 promotes the expansion of the latently infected B cell pool in vivo and may do so in part through inhibition of terminal differentiation. These findings provide new insight into mechanisms by which viral ncRNAs promote in vivo colonization and further and provide further evidence of the inherent tumorigenic risks associated with gammaherpesvirus manipulation of B cell differentiation. IMPORTANCE The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68, employ numerous strategies to colonize the host, including expression of noncoding RNAs (ncRNAs). As the first viral ncRNAs ever identified, EBV-encoded RNA 1 and 2 (EBER1 and EBER2) have been investigated extensively for decades; however, their specific in vivo functions remain largely unknown. Work here reveals that an EBV EBER2 variant highly associated with B cell lymphoma promoted a significantly increased expansion of the infected B cell pool in vivo, which coincided with altered B cell differentiation. Mutation of the 5 nucleotides that define this EBER2 variant resulted in the loss of B cell expansion and normal B cell differentiation. These findings provide new insight into the mechanisms by which EBV manipulates B cells in vivo to retain infected cells in the high-risk B cell differentiation pathway where they are poised for tumorigenesis.
Assuntos
Infecções por Vírus Epstein-Barr , Gammaherpesvirinae , Herpesvirus Humano 8 , Rhadinovirus , Animais , Infecções por Vírus Epstein-Barr/genética , Gammaherpesvirinae/genética , Herpesvirus Humano 4/fisiologia , Herpesvirus Humano 8/genética , Humanos , Camundongos , Nucleotídeos , Polimorfismo Genético , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , RNA Viral , Rhadinovirus/genética , Latência Viral/genéticaRESUMO
An integral feature of gammaherpesvirus infections is the ability to establish lifelong latency in B cells. During latency, the viral genome is maintained as an extrachomosomal episome, with stable maintenance in dividing cells mediated by the viral proteins Epstein-Barr nuclear antigen 1 (EBNA-1) for Epstein-Barr virus and latency-associated nuclear antigen (LANA) for Kaposi's sarcoma-associated herpesvirus. It is believed that the expression of episome maintenance proteins is turned off in the predominant long-term latency reservoir of resting memory B cells, suggesting that chronic gammaherpesvirus infection is primarily dormant. However, the kinetics of LANA/EBNA-1 expression in individual B-cell subsets throughout a course of infection has not been examined. The infection of mice with murine gammaherpesvirus 68 (MHV68, gammaHV68) provides a model to determine the specific cellular and molecular events that occur in vivo during lifelong gammaherpesvirus latency. In work described here, we make use of a heterologously expressed enzymatic marker to define the types of B cells that express the LANA homolog (mLANA) during chronic MHV68 infection. Our data demonstrate that mLANA is expressed in a stable fraction of B cells throughout chronic infection, with a prominent peak at 28 days. The expression of mLANA was detected in naïve follicular B cells, germinal-center B cells, and memory B cells throughout infection, with germinal-center and memory B cells accounting for more than 80% of the mLANA-expressing cells during the maintenance phase of latency. These findings suggest that the maintenance phase of latency is an active process that involves the ongoing proliferation or reseeding of latently infected memory B cells.