RESUMO
Gammaherpesviruses are lymphotropic viruses that are associated with the development of lymphoproliferative diseases, lymphomas, as well as other nonlymphoid cancers. Most known gammaherpesviruses establish latency in B lymphocytes. Research on Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68/γHV68/MHV4) has revealed a complex relationship between virus latency and the stage of B cell differentiation. Available data support a model in which gammaherpesvirus infection drives B cell proliferation and differentiation. In general, the characterized gammaherpesviruses exhibit a very narrow host tropism, which has severely limited studies on the human gammaherpesviruses EBV and Kaposi's sarcoma-associated herpesvirus. As such, there has been significant interest in developing animal models in which the pathogenesis of gammaherpesviruses can be characterized. MHV68 represents a unique model to define the effects of chronic viral infection on the antiviral immune response.
Assuntos
Gammaherpesvirinae/fisiologia , Animais , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Especificidade de Hospedeiro , Humanos , Camundongos , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/virologiaRESUMO
One of the defining characteristics of the B cell receptor (BCR) is the extensive diversity in the repertoire of immunoglobulin genes that make up the BCR, resulting in broad range of specificity. Gammaherpesviruses are B lymphotropic viruses that establish life-long infection in B cells, and although the B cell receptor plays a central role in B cell biology, very little is known about the immunoglobulin repertoire of gammaherpesvirus infected cells. To begin to characterize the Ig genes expressed by murine gammaherpesvirus 68 (MHV68) infected cells, we utilized single cell sorting to sequence and clone the Ig variable regions of infected germinal center (GC) B cells and plasma cells. We show that MHV68 infection is biased towards cells that express the Igλ light chain along with a single heavy chain variable gene, IGHV10-1*01. This population arises through clonal expansion but is not viral antigen specific. Furthermore, we show that class-switching in MHV68 infected cells differs from that of uninfected cells. Fewer infected GC B cells are class-switched compared to uninfected GC B cells, while more infected plasma cells are class-switched compared to uninfected plasma cells. Additionally, although they are germinal center derived, the majority of class switched plasma cells display no somatic hypermutation regardless of infection status. Taken together, these data indicate that selection of infected B cells with a specific BCR, as well as virus mediated manipulation of class switching and somatic hypermutation, are critical aspects in establishing life-long gammaherpesvirus infection.
Assuntos
Linfócitos B/imunologia , Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/veterinária , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Cadeias lambda de Imunoglobulina/imunologia , Doenças dos Roedores/imunologia , Animais , Linfócitos B/virologia , Feminino , Gammaherpesvirinae/genética , Centro Germinativo/imunologia , Centro Germinativo/virologia , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Plasmócitos/imunologia , Plasmócitos/virologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Doenças dos Roedores/genética , Doenças dos Roedores/virologiaRESUMO
Gammaherpesviruses have evolved various strategies to take advantage of host cellular factors or signaling pathways to establish a lifelong latent infection. Like the human gammaherpesvirus Epstein-Barr virus, murine gammaherpesvirus 68 (MHV68) establishes and maintains latency in the memory B cells during infection of laboratory mice. We have previously shown that MHV68 can immortalize fetal liver-derived B cells that induce lymphomas when injected into immunodeficient mice. Here we identify interleukin 16 (IL16) as a most abundantly expressed cytokine in MHV68-immortalized B cells and show that MHV68 infection elevates IL16 expression. IL16 is not important for MHV68 lytic infection but plays a critical role in MHV68 reactivation from latency. IL16 deficiency increases MHV68 lytic gene expression in MHV68-immortalized B cells and enhances reactivation from splenic latency. Correlatively, IL16 deficiency increases the frequency of MHV68-infected plasma cells that can be attributed to enhanced MHV68 reactivation. Furthermore, similar to TPA-mediated lytic replication of Kaposi's sarcoma-associated herpesvirus, IL16 deficiency markedly induces Tyr705 STAT3 de-phosphorylation and elevates p21 expression, which can be counteracted by the tyrosine phosphatase inhibitor orthovanadate. Importantly, orthovanadate strongly blocks MHV68 lytic gene expression mediated by IL16 deficiency. These data demonstrate that virus-induced IL16 does not directly participate in MHV68 lytic replication, but rather inhibits virus reactivation to facilitate latent infection, in part through the STAT3-p21 axis.
Assuntos
Infecções por Herpesviridae/metabolismo , Interleucina-16/metabolismo , Infecções Tumorais por Vírus/metabolismo , Ativação Viral/fisiologia , Latência Viral/fisiologia , Animais , Linfócitos B/virologia , Infecções por Herpesviridae/imunologia , Interleucina-16/imunologia , Linfoma/virologia , Camundongos , Rhadinovirus/imunologia , Rhadinovirus/metabolismoRESUMO
Caspase-8 (Casp8)-mediated signaling triggers extrinsic apoptosis while suppressing receptor-interacting protein kinase (RIPK) 3-dependent necroptosis. Although Casp8 is dispensable for the development of innate and adaptive immune compartments in mice, the importance of this proapoptotic protease in the orchestration of immune response to pathogens remains to be fully explored. In this study, Casp8-/-Ripk3-/- C57BL/6 mice show robust innate and adaptive immune responses to the natural mouse pathogen, murine CMV. When young, these mice lack lpr-like lymphoid hyperplasia and accumulation of either B220 + CD3+ or B220-CD3+CD4+ and CD8+ T cells with increased numbers of immature myeloid cells that are evident in older mice. Dendritic cell activation and cytokine production drive both NK and T cell responses to control viral infection in these mice, suggesting that Casp8 is dispensable to the generation of antiviral host defense. Curiously, NK and T cell expansion is amplified, with greater numbers observed by 7 d postinfection compared with either Casp8+/-Ripk3-/- or wild type (Casp8+/+Ripk3+/+ ) littermate controls. Casp8 and RIPK3 are natural targets of virus-encoded cell death suppressors that prevent infected cell apoptosis and necroptosis, respectively. It is clear from the current studies that the initiation of innate immunity and the execution of cytotoxic lymphocyte functions are all preserved despite the absence of Casp8 in responding cells. Thus, Casp8 and RIPK3 signaling is completely dispensable to the generation of immunity against this natural herpesvirus infection, although the pathways driven by these initiators serve as a crucial first line for host defense within virus-infected cells.
Assuntos
Caspase 8/genética , Células Dendríticas/imunologia , Infecções por Herpesviridae/imunologia , Muromegalovirus/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Imunidade Adaptativa , Animais , Antígenos Virais/imunologia , Apoptose , Células Dendríticas/virologia , Humanos , Imunidade Inata , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de SinaisRESUMO
Murine gammaherpesvirus 68 (MHV68) infection of laboratory strains of mice has provided a tractable small animal model for dissecting gammaherpesvirus pathogenesis. The MHV68 latency associated antigen M2 promotes viral latency establishment in germinal center (GC) B cells and plays an important role in virus infection of plasma cells (PCs), which is linked to virus reactivation. More recently, M2 has been highlighted as a potent immunomodulatory molecule capable of hindering both cell-mediated and humoral immunity to MHV68 infection and subsequent challenges. M2 expression in B cells results in activation of B cell receptor signaling pathways that promote proliferation, differentiation, and cytokine production-a hallmark of gammaherpesviruses. In this study, we utilized an adoptive transfer model to explore the biological consequence of M2 expression in activated B cells in vivo. Secondly, we engineered and validated two independent MHV68 M2 reporter viruses that track M2 protein expression in latently infected B cells during infection. Here we demonstrate that upon adoptive transfer into naive mice, M2 expression promotes activated primary B cells to competitively establish residency in the spleen as either a GC B cell or a PC, most notably in the absence of an ongoing GC reaction. Moreover, M2 antigen drives robust PC differentiation and IL10 production in vivo in the absence of other viral factors. Lastly, we confirm that M2 expression during MHV68 infection is localized to the GC compartment, which is a long term latency reservoir for gammaherpesviruses. Overall, these observations are consistent with, and extend upon previous reports of M2 function in B cells and within the context of MHV68 infection. Moreover, this work provides support for a model by which M2-driven dysregulation of B cell function compromises multiple aspects of antiviral immunity to achieve persistence within the infected host.
Assuntos
Linfócitos B/imunologia , Linfócitos B/virologia , Infecções por Herpesviridae/imunologia , Ativação Linfocitária/imunologia , Transferência Adotiva , Animais , Diferenciação Celular/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Centro Germinativo/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Rhadinovirus/imunologia , Baço/imunologia , Proteínas Virais , Latência Viral/imunologiaRESUMO
T: Activator protein 1 (AP-1) is a transcription factor that recognizes two versions of a 7-base pair response element, either 5Î- GAG CA-3Î or 5Î- GAG CA-3Î (where M = 5-methylcytosine). These two elements share the feature that 5-methylcytosine and thymine both have a methyl group in the same position, 5-carbon of the pyrimidine, so each of them has two methyl groups at nucleotide positions 1 and 5 from the 5Î end, resulting in four methyl groups symmetrically positioned in duplex DNA. Epstein-Barr Virus Zta is a key transcriptional regulator of the viral lytic cycle that is homologous to AP-1. Zta recognizes several methylated Zta-response elements, including meZRE1 (5Î- GAG C A-3Î) and meZRE2 (5Î- GAG G A-3Î), where a methylated cytosine occupies one of the inner thymine residues corresponding to the AP-1 element, resulting in the four spatially equivalent methyl groups. Here, we study how AP-1 and Zta recognize these methyl groups within their cognate response elements. These methyl groups are in van der Waals contact with a conserved di-alanine in AP-1 dimer (Ala265 and Ala266 in Jun), or with the corresponding Zta residues Ala185 and Ser186 (via its side chain carbon Cß atom). Furthermore, the two ZRE elements differ at base pair 6 (C:G versus G:C), forming a pseudo-symmetric sequence (meZRE1) or an asymmetric sequence (meZRE2). In vitro DNA binding assays suggest that Zta has high affinity for all four sequences examined, whereas AP-1 has considerably reduced affinity for the asymmetric sequence (meZRE2). We ascribe this difference to Zta Ser186 (a unique residue for Zta) whose side chain hydroxyl oxygen atom interacts with the two half sites differently, whereas the corresponding Ala266 of AP-1 Jun protein lacks such flexibility. Our analyses demonstrate a novel mechanism of 5mC/T recognition in a methylation-dependent, spatial and sequence-specific approach by basic leucine-zipper transcriptional factors.
Assuntos
Metilação de DNA , Proteínas Proto-Oncogênicas c-jun/química , Elementos de Resposta , Transativadores/química , 5-Metilcitosina/química , Pareamento de Bases , Sítios de Ligação , DNA/química , DNA/metabolismo , Humanos , Ligação Proteica , Multimerização Proteica , Proteínas Proto-Oncogênicas c-jun/metabolismo , Timina/química , Transativadores/metabolismo , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismoRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma and multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50 RTA is a known transactivator of multiple viral genes, allowing it to control the switch between latency and virus replication. We report here the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial), 3T12 (mouse fibroblast), and RAW 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 to 10 residues at the amino terminus of the protein. We show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation. IMPORTANCE: Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma and multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV-infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication and is conserved among all known gammaherpesviruses. We show here for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of several isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression, leading to the efficient production of mature virions.
Assuntos
Processamento Alternativo , Herpesvirus Humano 8/genética , Proteínas Imediatamente Precoces/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Transativadores/genética , Ativação Transcricional , Animais , Linfócitos B/metabolismo , Linfócitos B/virologia , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Fibroblastos/metabolismo , Fibroblastos/virologia , Células HEK293 , Herpesvirus Humano 8/metabolismo , Humanos , Proteínas Imediatamente Precoces/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Sarcoma de Kaposi/virologia , Transativadores/metabolismo , Células Vero , Latência Viral/genética , Replicação Viral/genéticaRESUMO
The human gammaherpesviruses take advantage of normal B cell differentiation pathways to establish life-long infection in memory B cells. Murine gammaherpesvirus 68 (MHV68) infection of laboratory strains of mice also leads to life-long infection in memory B cells. To gain access to the memory B cell population, MHV68 infected B cells pass through the germinal center reaction during the onset of latency and require signals from T follicular helper (TFH) cells for proliferation. Interleukin 21 (IL-21), one of the secreted factors produced by TFH cells, plays an important role in both the maintenance of the germinal center response as well as in the generation of long-lived plasma cells. Using IL-21R deficient mice, we show that IL-21 signaling is required for efficient establishment of MHV68 infection. In the absence of IL-21 signaling, fewer infected splenocytes are able to gain access to either the germinal center B cell population or the plasma cell population--the latter being a major site of MHV68 reactivation. Furthermore, the germinal center B cell population in IL-21R(-/-) mice is skewed towards the non-proliferating centrocyte phenotype, resulting in reduced expansion of infected B cells. Additionally, the reduced frequency of infected plasma cells results in a significant reduction in the frequency of splenocytes capable of reactivating virus. This defect in establishment of MHV68 infection is intrinsic to B cells, as MHV68 preferentially establishes infection in IL-21R sufficient B cells in mixed bone marrow chimeric mice. Taken together, these data indicate that IL-21 signaling plays multiple roles during establishment of MHV68 infection, and identify IL-21 as a critical TFH cell-derived factor for efficient establishment of gammaherpesvirus B cell latency.
Assuntos
Linfócitos B/virologia , Infecções por Herpesviridae/imunologia , Interleucinas/imunologia , Rhadinovirus/fisiologia , Transdução de Sinais , Latência Viral/imunologia , Animais , Linfócitos B/imunologia , Feminino , Citometria de Fluxo , Centro Germinativo/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Immunity to non-cerebral severe malaria is estimated to occur within 1-2 infections in areas of endemic transmission for Plasmodium falciparum. Yet, nearly 20% of infected children die annually as a result of severe malaria. Multiple risk factors are postulated to exacerbate malarial disease, one being co-infections with other pathogens. Children living in Sub-Saharan Africa are seropositive for Epstein Barr Virus (EBV) by the age of 6 months. This timing overlaps with the waning of protective maternal antibodies and susceptibility to primary Plasmodium infection. However, the impact of acute EBV infection on the generation of anti-malarial immunity is unknown. Using well established mouse models of infection, we show here that acute, but not latent murine gammaherpesvirus 68 (MHV68) infection suppresses the anti-malarial humoral response to a secondary malaria infection. Importantly, this resulted in the transformation of a non-lethal P. yoelii XNL infection into a lethal one; an outcome that is correlated with a defect in the maintenance of germinal center B cells and T follicular helper (Tfh) cells in the spleen. Furthermore, we have identified the MHV68 M2 protein as an important virus encoded protein that can: (i) suppress anti-MHV68 humoral responses during acute MHV68 infection; and (ii) plays a critical role in the observed suppression of anti-malarial humoral responses in the setting of co-infection. Notably, co-infection with an M2-null mutant MHV68 eliminates lethality of P. yoelii XNL. Collectively, our data demonstrates that an acute gammaherpesvirus infection can negatively impact the development of an anti-malarial immune response. This suggests that acute infection with EBV should be investigated as a risk factor for non-cerebral severe malaria in young children living in areas endemic for Plasmodium transmission.
Assuntos
Coinfecção/imunologia , Infecções por Herpesviridae/imunologia , Herpesviridae/imunologia , Imunidade Humoral/imunologia , Malária/imunologia , Malária/virologia , Animais , Diferenciação Celular/imunologia , Feminino , Camundongos Endogâmicos C57BL , Ativação Viral/imunologia , Latência Viral/imunologiaRESUMO
The pronecrotic kinase, receptor interacting protein (RIP1, also called RIPK1) mediates programmed necrosis and, together with its partner, RIP3 (RIPK3), drives midgestational death of caspase 8 (Casp8)-deficient embryos. RIP1 controls a second vital step in mammalian development immediately after birth, the mechanism of which remains unresolved. Rip1(-/-) mice display perinatal lethality, accompanied by gross immune system abnormalities. Here we show that RIP1 K45A (kinase dead) knockin mice develop normally into adulthood, indicating that development does not require RIP1 kinase activity. In the face of complete RIP1 deficiency, cells develop sensitivity to RIP3-mixed lineage kinase domain-like-mediated necroptosis as well as to Casp8-mediated apoptosis activated by diverse innate immune stimuli (e.g., TNF, IFN, double-stranded RNA). When either RIP3 or Casp8 is disrupted in combination with RIP1, the resulting double knockout mice exhibit slightly prolonged survival over RIP1-deficient animals. Surprisingly, triple knockout mice with combined RIP1, RIP3, and Casp8 deficiency develop into viable and fertile adults, with the capacity to produce normal levels of myeloid and lymphoid lineage cells. Despite the combined deficiency, these mice sustain a functional immune system that responds robustly to viral challenge. A single allele of Rip3 is tolerated in Rip1(-/-)Casp8(-/-)Rip3(+/-) mice, contrasting the need to eliminate both alleles of either Rip1 or Rip3 to rescue midgestational death of Casp8-deficient mice. These observations reveal a vital kinase-independent role for RIP1 in preventing pronecrotic as well as proapoptotic signaling events associated with life-threatening innate immune activation at the time of mammalian parturition.
Assuntos
Apoptose/imunologia , Proteínas Ativadoras de GTPase/imunologia , Imunidade Inata/imunologia , Necrose/imunologia , Parto/imunologia , Transdução de Sinais/imunologia , Animais , Caspase 8/imunologia , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Técnicas de Introdução de Genes , Immunoblotting , Camundongos , Camundongos Knockout , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologiaRESUMO
X linked lymphoproliferative disease (XLP) is an inherited immunodeficiency resulting from mutations in the gene encoding the slam associated protein (SAP). One of the defining characteristics of XLP is extreme susceptibility to infection with Epstein-Barr virus (EBV), a gammaherpesvirus belonging to the genus Lymphocryptovirus, often resulting in fatal infectious mononucleosis (FIM). However, infection of SAP deficient mice with the related Murine gammaherpesvirus 68 (MHV68), a gammaherpesvirus in the genus Rhadinovirus, does not recapitulate XLP. Here we show that MHV68 inefficiently establishes latency in B cells in SAP deficient mice due to insufficient CD4 T cell help during the germinal center response. Although MHV68 infected B cells can be found in SAP-deficient mice, significantly fewer of these cells had a germinal center phenotype compared to SAP-sufficient mice. Furthermore, we show that infected germinal center B cells in SAP-deficient mice fail to proliferate. This failure to proliferate resulted in significantly lower viral loads, and likely accounts for the inability of MHV68 to induce a FIM-like syndrome. Finally, inhibiting differentiation of T follicular helper (TFH) cells in SAP-sufficient C57Bl/6 mice resulted in decreased B cell latency, and the magnitude of the TFH response directly correlated with the level of infection in B cells. This requirement for CD4 T cell help during the germinal center reaction by MHV68 is in contrast with EBV, which is thought to be capable of bypassing this requirement by expressing viral proteins that mimic signals provided by TFH cells. In conclusion, the outcome of MHV68 infection in mice in the setting of loss of SAP function is distinct from that observed in SAP-deficient patients infected with EBV, and may identify a fundamental difference between the strategies employed by the rhadinoviruses and lymphocryptoviruses to expand B cell latency during the early phase of infection.
Assuntos
Linfócitos B/fisiologia , Linfócitos B/virologia , Linfócitos T CD4-Positivos/fisiologia , Proliferação de Células , Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/imunologia , Latência Viral , Animais , Linfócitos T CD4-Positivos/virologia , Centro Germinativo/imunologia , Centro Germinativo/virologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organismos Geneticamente Modificados , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Latência Viral/imunologiaRESUMO
Reactivation of the gammaherpesviruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) from latently infected B cells has been linked to plasma cell differentiation. We have previously shown that the MHV68 M2 protein is important for virus reactivation from B cells and, when expressed alone in primary murine B cells, can drive B cell differentiation towards a pre-plasma cell phenotype. In addition, expression of M2 in primary murine B cells leads to secretion of high levels of IL-10 along with enhanced proliferation and survival. Furthermore, the absence of M2 in vivo leads to a defect in the appearance of MHV68 infected plasma cells in the spleen at the peak of MHV68 latency. Here, employing an inducible B cell expression system, we have determined that M2 activates the NFAT pathway in a Src kinase-dependent manner--leading to induction of the plasma cell-associated transcription factor, Interferon Regulatory Factor-4 (IRF4). Furthermore, we show that expression of IRF4 alone in a B cell line up-regulates IL-10 expression in culture supernatants, revealing a novel role for IRF4 in B cell induced IL-10. Consistent with the latter observation, we show that IRF4 can regulate the IL-10 promoter in B cells. In primary murine B cells, addition of cyclosporine (CsA) resulted in a significant decrease in M2-induced IL-10 levels as well as IRF4 expression, emphasizing the importance of the NFAT pathway in M2- -mediated induction of IL-10. Together, these studies argue in favor of a model wherein M2 activation of the NFAT pathway initiates events leading to increased levels of IRF4--a key player in plasma cell differentiation--which in turn triggers IL-10 expression. In the context of previous findings, the data presented here provides insights into how M2 facilitates plasma cell differentiation and subsequent virus reactivation.
Assuntos
Linfócitos B/imunologia , Regulação da Expressão Gênica/imunologia , Fatores Reguladores de Interferon/imunologia , Interleucina-10/imunologia , Modelos Imunológicos , Fatores de Transcrição NFATC/imunologia , Rhadinovirus/fisiologia , Proteínas Virais/imunologia , Animais , Linfócitos B/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/patologia , Fatores Reguladores de Interferon/genética , Interleucina-10/genética , Ativação Linfocitária , Camundongos , Camundongos Mutantes , Fatores de Transcrição NFATC/genética , Proteínas Virais/genética , Latência Viral/genética , Latência Viral/imunologiaRESUMO
MHV68 is a murine gammaherpesvirus that infects laboratory mice and thus provides a tractable small animal model for characterizing critical aspects of gammaherpesvirus pathogenesis. Having evolved with their natural host, herpesviruses encode numerous gene products that are involved in modulating host immune responses to facilitate the establishment and maintenance of lifelong chronic infection. One such protein, MHV68 M1, is a secreted protein that has no known homologs, but has been shown to play a critical role in controlling virus reactivation from latently infected macrophages. We have previous demonstrated that M1 drives the activation and expansion of Vß4+ CD8+ T cells, which are thought to be involved in controlling MHV68 reactivation through the secretion of interferon gamma. The mechanism of action and regulation of M1 expression are poorly understood. To gain insights into the function of M1, we set out to evaluate the site of expression and transcriptional regulation of the M1 gene. Here, using a recombinant virus expressing a fluorescent protein driven by the M1 gene promoter, we identify plasma cells as the major cell type expressing M1 at the peak of infection in the spleen. In addition, we show that M1 gene transcription is regulated by both the essential viral immediate-early transcriptional activator Rta and cellular interferon regulatory factor 4 (IRF4), which together potently synergize to drive M1 gene expression. Finally, we show that IRF4, a cellular transcription factor essential for plasma cell differentiation, can directly interact with Rta. The latter observation raises the possibility that the interaction of Rta and IRF4 may be involved in regulating a number of viral and cellular genes during MHV68 reactivation linked to plasma cell differentiation.
Assuntos
Infecções por Herpesviridae/metabolismo , Plasmócitos/virologia , Superantígenos/metabolismo , Proteínas Virais/metabolismo , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Citometria de Fluxo , Gammaherpesvirinae , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/imunologia , Interações Hospedeiro-Parasita , Proteínas Imediatamente Precoces , Imunoprecipitação , Fatores Reguladores de Interferon , Camundongos , Camundongos Endogâmicos C57BL , Plasmócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superantígenos/genética , Proteínas Virais/genética , Ativação Viral/fisiologia , Latência Viral/fisiologiaRESUMO
UNLABELLED: The essential immediate early transcriptional activator RTA, encoded by gene 50, is conserved among all characterized gammaherpesviruses. Analyses of a recombinant murine gammaherpesvirus 68 (MHV68) lacking both of the known gene 50 promoters (G50DblKo) revealed that this mutant retained the ability to replicate in the simian kidney epithelial cell line Vero but not in permissive murine fibroblasts following low-multiplicity infection. However, G50DblKo replication in permissive fibroblasts was partially rescued by high-multiplicity infection. In addition, replication of the G50DblKo virus was rescued by growth on mouse embryonic fibroblasts (MEFs) isolated from IFN-α/ßR-/- mice, while growth on Vero cells was suppressed by the addition of alpha interferon (IFN-α). 5' rapid amplification of cDNA ends (RACE) analyses of RNAs prepared from G50DblKo and wild-type MHV68-infected murine macrophages identified three novel gene 50 transcripts initiating from 2 transcription initiation sites located upstream of the currently defined proximal and distal gene 50 promoters. In transient promoter assays, neither of the newly identified gene 50 promoters exhibited sensitivity to IFN-α treatment. Furthermore, in a single-step growth analysis RTA levels were higher at early times postinfection with the G50DblKo mutant than with wild-type virus but ultimately fell below the levels of RTA expressed by wild-type virus at later times in infection. Infection of mice with the MHV68 G50DblKo virus demonstrated that this mutant virus was able to establish latency in the spleen and peritoneal exudate cells (PECs) of C57BL/6 mice with about 1/10 the efficiency of wild-type virus or marker rescue virus. However, despite the ability to establish latency, the G50DblKo virus mutant was severely impaired in its ability to reactivate from either latently infected splenocytes or PECs. Consistent with the ability to rescue replication of the G50DblKo mutant by growth on type I interferon receptor null MEFs, infection of IFN-α/ßR-/- mice with the G50DblKo mutant virus demonstrated partial rescue of (i) acute virus replication in the lungs, (ii) establishment of latency, and (iii) reactivation from latency. The identification of additional gene 50/RTA transcripts highlights the complex mechanisms involved in controlling expression of RTA, likely reflecting time-dependent and/or cell-specific roles of different gene 50 promoters in controlling virus replication. Furthermore, the newly identified gene 50 transcripts may also act as negative regulators that modulate RTA expression. IMPORTANCE: The viral transcription factor RTA, encoded by open reading frame 50 (Orf50), is well conserved among all known gammaherpesviruses and is essential for both virus replication and reactivation from latently infected cells. Previous studies have shown that regulation of gene 50 transcription is complex. The studies reported here describe the presence of additional alternatively initiated, spliced transcripts that encode RTA. Understanding how expression of this essential viral gene product is regulated may identify new strategies for interfering with infection in the setting of gammaherpesvirus-induced diseases.
Assuntos
Regulação Viral da Expressão Gênica , Rhadinovirus/genética , Transativadores/biossíntese , Transativadores/genética , Transcrição Gênica , Animais , Células Cultivadas , Chlorocebus aethiops , Feminino , Fibroblastos/virologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Rhadinovirus/fisiologia , Ativação Viral , Latência Viral , Replicação ViralRESUMO
UNLABELLED: Gammaherpesviruses display tropism for B cells and, like all known herpesviruses, exhibit distinct lytic and latent life cycles. One well-established observation among members of the gammaherpesvirus family is the link between viral reactivation from latently infected B cells and plasma cell differentiation. Importantly, a number of studies have identified a potential role for a CREB/ATF family member, X-box binding protein 1 (XBP-1), in trans-activating the immediate early BZLF-1 or BRLF1/gene 50 promoters of Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), respectively. XBP-1 is required for the unfolded protein response and has been identified as a critical transcription factor in plasma cells. Here, we demonstrate that XBP-1 is capable of trans-activating the murine gammaherpesvirus 68 (MHV68) RTA promoter in vitro, consistent with previous observations for EBV and KSHV. However, we show that in vivo there does not appear to be a requirement for XBP-1 expression in B cells for virus reactivation. The MHV68 M2 gene product under some experimental conditions plays an important role in virus reactivation from B cells. M2 has been shown to drive B cell differentiation to plasma cells, as well as interleukin-10 (IL-10) production, both of which are dependent on M2 induction of interferon regulatory factor 4 (IRF4) expression. IRF4 is required for plasma cell differentiation, and consistent with a role for plasma cells in MHV68 reactivation from B cells, we show that IRF4 expression in B cells is required for efficient reactivation of MHV68 from splenocytes. Thus, the latter analyses are consistent with previous studies linking plasma cell differentiation to MHV68 reactivation from B cells. The apparent independence of MHV68 reactivation from XBP-1 expression in plasma cells may reflect redundancy among CREB/ATF family members or the involvement of other plasma cell-specific transcription factors. Regardless, these findings underscore the importance of in vivo studies in assessing the relevance of observations made in tissue culture models. IMPORTANCE: All known herpesviruses establish a chronic infection of their respective host, persisting for the life of the individual. A critical feature of these viruses is their ability to reactivate from a quiescent form of infection (latency) and generate progeny virus. In the case of gammaherpesviruses, which are associated with the development of lymphoproliferative disorders, including lymphomas, reactivation from latently infected B lymphocytes occurs upon terminal differentiation of these cells to plasma cells-the cell type that produces antibodies. A number of studies have linked a plasma cell transcription factor, XBP-1, to the induction of gammaherpesvirus reactivation, and we show here that indeed in tissue culture models this cellular transcription factor can trigger expression of the murine gammaherpesvirus gene involved in driving virus reactivation. However, surprisingly, when we examined the role of XBP-1 in the setting of infection of mice-using mice that lack a functional XBP-1 gene in B cells-we failed to observe a role for XBP-1 in virus reactivation. However, we show that another cellular factor essential for plasma cell differentiation, IRF4, is critical for virus reactivation. Thus, these studies point out the importance of studies in animal models to validate findings from studies carried out in cell lines passaged in vitro.
Assuntos
Linfócitos B/virologia , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/genética , Fatores Reguladores de Interferon/genética , Rhadinovirus/genética , Proteínas Virais/genética , Animais , Linfócitos B/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Interações Hospedeiro-Patógeno , Fatores Reguladores de Interferon/metabolismo , Camundongos , Plasmócitos/metabolismo , Plasmócitos/virologia , Regiões Promotoras Genéticas , Fatores de Transcrição de Fator Regulador X , Rhadinovirus/metabolismo , Transdução de Sinais , Baço/metabolismo , Baço/virologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Ativação Viral , Latência Viral , Proteína 1 de Ligação a X-BoxRESUMO
There has been extensive research regarding T cell recognition of Epstein-Barr virus-transformed cells; however, less is known regarding the recognition of B cells immortalized by gamma-2 herpesviruses. Here we show that B cells immortalized by murine gammaherpesvirus 68 (MHV-68, γHV-68) can be controlled by either CD4 or CD8 T cells in vivo. We present evidence for the direct recognition of infected B cells by CD4 and CD8 T cells. These data will help in the development of immunotherapeutic approaches combating gamma-2 herpesvirus-related disease.
Assuntos
Linfócitos B/imunologia , Linfócitos B/virologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Transformação Celular Viral , Rhadinovirus/patogenicidade , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The secreted M1 protein of murine gammaherpesvirus 68 (MHV68) promotes effector Vß4(+) CD8(+) T cell expansion to impact virus control and immune-mediated pathologies in C57BL/6 mice, but not BALB/c mice. We report a striking increase in the number of genome-positive, IgD(-) B cells during chronic infection of both mouse strains. This suggests a novel role for M1 in influencing long-term maintenance in a major latency reservoir irrespective of the degree of Vß4(+) CD8(+) T cell expansion.
Assuntos
Linfócitos B/virologia , Imunoglobulina D/imunologia , Rhadinovirus/imunologia , Rhadinovirus/patogenicidade , Proteínas Virais/metabolismo , Latência Viral , Animais , Linfócitos B/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Virais/imunologiaRESUMO
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
Several studies have previously defined host-derived signaling events capable of driving lytic gammaherpesvirus replication or enhancing immediate-early viral gene expression. Yet signaling pathways that regulate later stages of the productive gammaherpesvirus replication cycle are still poorly defined. In this study, we utilized a mass spectrometric approach to identify c-Jun as an abundant cellular phosphoprotein present in late stages of lytic murine gammaherpesvirus 68 (MHV68) infection. Kinetically, c-Jun phosphorylation was enhanced as infection progressed, and this correlated with enhanced phosphorylation of the c-Jun amino-terminal kinases JNK1 and JNK2 and activation of AP-1 transcription. These events were dependent on progression beyond viral immediate-early gene expression, but not dependent on viral DNA replication. Both pharmacologic and dominant-negative blockade of JNK1/2 activity inhibited viral replication, and this correlated with inhibition of viral DNA synthesis and reduced viral gene expression. These data suggest a model in which MHV68 by necessity amplifies and usurps JNK/c-Jun signaling as infection progresses in order to facilitate late stages of the MHV68 lytic infection cycle.
Assuntos
Gammaherpesvirinae/fisiologia , MAP Quinase Quinase 4/metabolismo , Transdução de Sinais , Replicação Viral , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Espectrometria de Massas em Tandem , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Human gammaherpesviruses are associated with the development of lymphoproliferative diseases and B cell lymphomas, particularly in immunosuppressed hosts. Understanding the molecular mechanisms by which human gammaherpesviruses cause disease is hampered by the lack of convenient small animal models to study them. However, infection of laboratory strains of mice with the rodent virus murine gammaherpesvirus 68 (MHV68) has been useful in gaining insights into how gammaherpesviruses contribute to the genesis and progression of lymphoproliferative lesions. In this report we make the novel observation that MHV68 infection of murine day 15 fetal liver cells results in their immortalization and differentiation into B plasmablasts that can be propagated indefinitely in vitro, and can establish metastasizing lymphomas in mice lacking normal immune competence. The phenotype of the MHV68 immortalized B cell lines is similar to that observed in lymphomas caused by KSHV and resembles the favored phenotype observed during MHV68 infection in vivo. All established cell lines maintained the MHV68 genome, with limited viral gene expression and little or no detectable virus production - although virus reactivation could be induced upon crosslinking surface Ig. Notably, transcription of the genes encoding the MHV68 viral cyclin D homolog (v-cyclin) and the homolog of the KSHV latency-associated nuclear antigen (LANA), both of which are conserved among characterized γ2-herpesviruses, could consistently be detected in the established B cell lines. Furthermore, we show that the v-cyclin and LANA homologs are required for MHV68 immortalization of murine B cells. In contrast the M2 gene, which is unique to MHV68 and plays a role in latency and virus reactivation in vivo, was dispensable for B cell immortalization. This new model of gammaherpesvirus-driven B cell immortalization and differentiation in a small animal model establishes an experimental system for detailed investigation of the role of gammaherpesvirus gene products and host responses in the genesis and progression of gammaherpesvirus-associated lymphomas, and presents a convenient system to evaluate therapeutic modalities.