RESUMEN
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.
Asunto(s)
Empalme Alternativo , Herpesvirus Humano 8 , Transcriptoma , Herpesvirus Humano 8/genética , Humanos , Transcriptoma/genética , Transcripción Genética , Regulación Viral de la Expresión Génica , Sistemas de Lectura Abierta/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Sarcoma de Kaposi/virología , Sarcoma de Kaposi/genética , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
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.
Asunto(s)
Linfocitos B , Gammaherpesvirinae , Centro Germinal , Infecciones por Herpesviridae , MicroARNs , Proteína EWS de Unión a ARN , Infecciones Tumorales por Virus , Animales , Linfocitos B/inmunología , Linfocitos B/virología , Gammaherpesvirinae/genética , Gammaherpesvirinae/fisiología , Eliminación de Gen , Centro Germinal/inmunología , Centro Germinal/virología , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/inmunología , Infecciones Tumorales por Virus/virología , Latencia del VirusRESUMEN
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.
Asunto(s)
Linfocitos B , Gammaherpesvirinae , Infecciones por Herpesviridae , Infección Persistente , Animales , Ratones , Linfocitos B/enzimología , Linfocitos B/metabolismo , Linfocitos B/virología , Proteínas Cullin/metabolismo , Gammaherpesvirinae/fisiología , Centro Germinal/citología , Centro Germinal/virología , Infecciones por Herpesviridae/enzimología , Infecciones por Herpesviridae/virología , Infección Persistente/enzimología , Infección Persistente/virología , Ubiquitinas/metabolismo , Latencia del VirusRESUMEN
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.
Asunto(s)
Gammaherpesvirinae/genética , ARN no Traducido , ARN Viral , Liberación del Virus/genética , Animales , Células Cultivadas , Infecciones por Herpesviridae/virología , Ratones , Conformación de Ácido Nucleico , ARN no Traducido/química , ARN no Traducido/genética , ARN no Traducido/fisiología , ARN Viral/química , ARN Viral/genética , ARN Viral/fisiología , Bazo/citología , Bazo/virología , Latencia del Virus/genéticaRESUMEN
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.
Asunto(s)
Gammaherpesvirinae/fisiología , Infecciones por Herpesviridae/metabolismo , MicroARNs/genética , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/metabolismo , Animales , Regulación de la Expresión Génica , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/virología , Ratones , ARN Mensajero/genética , ARN Viral/genética , ARN Viral/metabolismo , Serina-Treonina Quinasas TOR/genética , Factores de Transcripción/genética , Replicación ViralRESUMEN
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.
Asunto(s)
Gammaherpesvirinae/genética , ARN/genética , Animales , Línea Celular , Regulación Viral de la Expresión Génica/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 8/genética , Humanos , Lymphocryptovirus/genética , Macaca mulatta , Masculino , ARN Circular , ARN Viral/genética , Rhadinovirus/genética , Virus de la Inmunodeficiencia de los Simios/genética , Latencia del Virus/genética , Replicación Viral/genéticaRESUMEN
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.
Asunto(s)
Gammaherpesvirinae/fisiología , Infecciones por Herpesviridae/virología , Pulmón/virología , Macrófagos/virología , Sistemas de Lectura Abierta , Bazo/virología , Proteínas Virales/metabolismo , Animales , Células de la Médula Ósea/citología , Células Cultivadas , Codón sin Sentido , ADN Recombinante/metabolismo , ADN Viral/metabolismo , Embrión de Mamíferos/citología , Gammaherpesvirinae/crecimiento & desarrollo , Gammaherpesvirinae/patogenicidad , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/patología , Pulmón/inmunología , Pulmón/patología , Macrófagos/inmunología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Filogenia , Bazo/inmunología , Bazo/patología , Carga Viral , Proteínas Virales/genética , Latencia del Virus , Replicación ViralRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1006843.].
RESUMEN
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.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Infecciones por Herpesviridae/virología , Herpesvirus Humano 8/metabolismo , Rhadinovirus/genética , Infecciones Tumorales por Virus/virología , Proteínas Virales/metabolismo , Latencia del Virus/fisiología , Animales , Modelos Animales de Enfermedad , Proteínas de Unión al GTP/genética , Herpesvirus Humano 8/genética , Humanos , Ratones , Neovascularización Patológica/virología , Proteínas Virales/genética , Latencia del Virus/inmunologíaRESUMEN
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.
Asunto(s)
Linfocitos B/virología , Gammaherpesvirinae/fisiología , Infecciones por Herpesviridae/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Infecciones Tumorales por Virus/inmunología , Latencia del Virus/inmunología , Animales , Apoptosis/inmunología , Linfocitos B/citología , Western Blotting , Diferenciación Celular/inmunología , Supervivencia Celular/inmunología , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos B/inmunologíaRESUMEN
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.
Asunto(s)
Infecciones por Caliciviridae/virología , Enterocitos/virología , Gastroenteritis/virología , Norovirus/patogenicidad , Animales , Antivirales/uso terapéutico , Infecciones por Caliciviridae/tratamiento farmacológico , Infecciones por Caliciviridae/microbiología , Técnicas de Cultivo de Célula , Interacciones Huésped-Patógeno , Humanos , Intestinos/microbiología , Intestinos/virología , Ratones , Norovirus/fisiología , Simbiosis , Tropismo Viral , Replicación ViralRESUMEN
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.
Asunto(s)
Antígenos Virales/genética , Antígenos Virales/fisiología , ADN/metabolismo , Gammaherpesvirinae/genética , Mutagénesis/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Dominios y Motivos de Interacción de Proteínas/genética , Animales , Antígenos Virales/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación Viral de la Expresión Génica , Células HEK293 , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/virología , Humanos , Ratones , Células 3T3 NIH , Proteínas Nucleares/química , Dominios y Motivos de Interacción de Proteínas/fisiología , Represión Psicológica , Secuencias Repetidas Terminales/genéticaRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Interacciones Huésped-Patógeno/inmunología , Quinasa I-kappa B/metabolismo , Rhadinovirus/fisiología , Replicación Viral , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Infecciones por Herpesviridae , Quinasa I-kappa B/inmunología , Ratones , Fosforilación , Activación Transcripcional , Infecciones Tumorales por VirusRESUMEN
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.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Gammaherpesvirinae , Herpesvirus Humano 8 , Rhadinovirus , Animales , Infecciones por Virus de Epstein-Barr/genética , Gammaherpesvirinae/genética , Herpesvirus Humano 4/fisiología , Herpesvirus Humano 8/genética , Humanos , Ratones , Nucleótidos , Polimorfismo Genético , ARN no Traducido/genética , ARN no Traducido/metabolismo , ARN Viral , Rhadinovirus/genética , Latencia del Virus/genéticaRESUMEN
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.
Asunto(s)
Antígenos Virales/metabolismo , Linfocitos B/virología , Infecciones por Herpesviridae/virología , Proteínas Nucleares/metabolismo , Células Precursoras de Linfocitos B/virología , Rhadinovirus/fisiología , Infecciones Tumorales por Virus/virología , Latencia del Virus , Animales , Linfocitos B/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Células Precursoras de Linfocitos B/inmunología , Rhadinovirus/patogenicidad , Bazo/inmunología , Bazo/virología , Activación Viral , Replicación ViralRESUMEN
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.
Asunto(s)
Antígenos Virales/biosíntesis , Linfocitos B/virología , Expresión Génica , Infecciones por Herpesviridae/virología , Proteínas Nucleares/biosíntesis , Rhadinovirus/patogenicidad , Latencia del Virus , Animales , Línea Celular , Ratones , Ratones Endogámicos C57BLRESUMEN
Viruses, like their metazoan hosts, have evolved to utilize intricate transcriptional mechanisms to generate a vast array of both coding and noncoding RNA transcripts. The resolution of specific noncoding RNA transcripts produced by viruses, particularly herpesviruses, presents a particularly difficult challenge due to their highly dense dsDNA genomes and their complex, overlapping, and context-dependent network of transcripts. While new long read sequencing platforms have facilitated the resolution of some noncoding transcripts from virus genomes, empirical molecular validation of transcripts from individual regions is essential. Herein, we demonstrate that the use of strand specific northern blots is essential for true validation of specific viral noncoding RNAs, and provide here a detailed molecular method for such an approach.
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Northern Blotting , Genes Sobrepuestos , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Viral/genética , Northern Blotting/métodos , Electroforesis en Gel de Poliacrilamida , Regulación Viral de la Expresión Génica , Genoma Viral , Herpesviridae/genética , Sistemas de Lectura Abierta , Virus/genéticaRESUMEN
Gammaherpesviruses are an important class of oncogenic pathogens that are exquisitely evolved to their respective hosts. As such, the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) do not naturally infect nonhuman primates or rodents. There is a clear need to fully explore mechanisms of gammaherpesvirus pathogenesis, host control, and immune evasion in the host. A gammaherpesvirus pathogen isolated from murid rodents was first reported in 1980; 40 years later, murine gammaherpesvirus 68 (MHV68, MuHV-4, γHV68) infection of laboratory mice is a well-established pathogenesis system recognized for its utility in applying state-of-the-art approaches to investigate virus-host interactions ranging from the whole host to the individual cell. Here, we highlight recent advancements in our understanding of the processes by which MHV68 colonizes the host and drives disease. Lessons that inform KSHV and EBV pathogenesis and provide future avenues for novel interventions against infection and virus-associated cancers are emphasized.
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Infecciones por Virus de Epstein-Barr , Gammaherpesvirinae , Infecciones por Herpesviridae , Rhadinovirus , Animales , Herpesvirus Humano 4 , Ratones , Latencia del VirusRESUMEN
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 (G305TTASSDTAITLIPR319) 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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Microbioma Gastrointestinal , Infecciones por Herpesviridae/microbiología , Animales , Antibacterianos/farmacología , Infecciones por Herpesviridae/tratamiento farmacológico , Infecciones por Herpesviridae/inmunología , Pulmón/efectos de los fármacos , Pulmón/patología , Linfocitos/inmunología , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/uso terapéutico , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapéutico , Serpinas/químicaRESUMEN
Murine gammaherpesvirus 68 (gammaHV68 or MHV68) is genetically related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), providing a useful system for in vivo studies of the virus-host relationship. To begin to address fundamental questions about the mechanisms of the establishment of gammaherpesvirus latency, we previously generated a replication-defective gammaHV68 lacking the expression of the single-stranded DNA binding protein encoded by orf6. In work presented here, we demonstrate that this mutant virus established a long-term infection in vivo that was molecularly identical to wild-type virus latency. Thus, despite the absence of an acute phase of lytic replication, the mutant virus established a chronic infection in which the viral genome (i) was maintained as an episome and (ii) expressed latency-associated, but not lytic replication-associated, genes. Macrophages purified from mice infected with the replication-defective virus harbored viral genome at a frequency that was nearly identical to that of wild-type gammaHV68; however, the frequency of B cells harboring viral genome was greatly reduced in the absence of lytic replication. Thus, this replication-defective gammaherpesvirus efficiently established in vivo infection in macrophages that was molecularly indistinguishable from wild-type virus latency. These data point to a critical role for lytic replication or reactivation in the establishment or maintenance of latent infection in B cells.