RESUMEN
Natural killer (NK) cells play a key role in innate immunity by detecting alterations in self and non-self ligands via paired NK cell receptors (NKRs). Despite identification of numerous NKR-ligand interactions, physiological ligands for the prototypical NK1.1 orphan receptor remain elusive. Here, we identify a viral ligand for the inhibitory and activating NKR-P1 (NK1.1) receptors. This murine cytomegalovirus (MCMV)-encoded protein, m12, restrains NK cell effector function by directly engaging the inhibitory NKR-P1B receptor. However, m12 also interacts with the activating NKR-P1A/C receptors to counterbalance m12 decoy function. Structural analyses reveal that m12 sequesters a large NKR-P1 surface area via a "polar claw" mechanism. Polymorphisms in, and ablation of, the viral m12 protein and host NKR-P1B/C alleles impact NK cell responses in vivo. Thus, we identify the long-sought foreign ligand for this key immunoregulatory NKR family and reveal how it controls the evolutionary balance of immune recognition during host-pathogen interplay.
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Células Asesinas Naturales/inmunología , Muromegalovirus/inmunología , Receptores de Células Asesinas Naturales/inmunología , Proteínas Virales/metabolismo , Animales , Antígenos Ly/metabolismo , Línea Celular , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Evasión Inmune , Inmunidad Innata , Ratones , Células 3T3 NIH , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismo , RatasRESUMEN
Viral infections during pregnancy are a considerable cause of adverse outcomes and birth defects, and the underlying mechanisms are poorly understood. Among those, cytomegalovirus (CMV) infection stands out as the most common intrauterine infection in humans, putatively causing early pregnancy loss. We employed murine CMV as a model to study the consequences of viral infection on pregnancy outcome and fertility maintenance. Even though pregnant mice successfully controlled CMV infection, we observed highly selective, strong infection of corpus luteum (CL) cells in their ovaries. High infection densities indicated complete failure of immune control in CL cells, resulting in progesterone insufficiency and pregnancy loss. An abundance of gap junctions, absence of vasculature, strong type I interferon (IFN) responses, and interaction of innate immune cells fully protected the ovarian follicles from viral infection. Our work provides fundamental insights into the effect of CMV infection on pregnancy loss and mechanisms protecting fertility.
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Cuerpo Lúteo/inmunología , Infecciones por Citomegalovirus/inmunología , Fertilidad/inmunología , Inmunidad Innata/inmunología , Animales , Cuerpo Lúteo/virología , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Femenino , Uniones Comunicantes/inmunología , Interferón Tipo I/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Embarazo , Progesterona/inmunologíaRESUMEN
In systemic lupus erythematosus (SLE) loss of immune tolerance, autoantibody production and immune complex deposition are required but not sufficient for organ damage1. How inflammatory signals are initiated and amplified in the setting of autoimmunity remains elusive. Here, we set out to dissect layers and hierarchies of autoimmune kidney inflammation in order to identify tissue-specific cellular hubs that amplify auto-inflammatory responses. Using high-resolution single-cell profiling of kidney immune and parenchymal cells, in combination with antibody blocking and genetic deficiency, we show that tissue-resident NKp46+ innate lymphoid cells (ILC) are crucial signal amplifiers of disease-associated macrophage expansion and epithelial cell injury in lupus nephritis, downstream of autoantibody production. NKp46 signaling in a distinct subset of ILC1 instructed an unconventional immune-regulatory transcriptional program, which included the expression of the myeloid cell growth factor CSF2. CSF2 production by NKp46+ ILC promoted the population expansion of monocyte-derived macrophages. Blockade of the NKp46 receptor (using the antibody mNCR1.152) or genetic deficiency of NKp46 abrogated epithelial cell injury. The same cellular and molecular patterns were operative in human lupus nephritis. Our data support that NKp46+ ILC1 promote parenchymal cell injury by granting monocyte-derived macrophages access to epithelial cell niches. NKp46 activation in ILC1 thus constitutes a previously unrecognized, critical tissue rheostat that amplifies organ damage in autoimmune hosts, with broad implications for inflammatory pathologies and therapies.
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Pro-inflammatory cytokines of a T helper-1-signature are known to promote insulin resistance (IR) in obesity, but the physiological role of this mechanism is unclear. It is also unknown whether and how viral infection induces loss of glycemic control in subjects at risk for developing diabetes mellitus type 2 (DM2). We have found in mice and humans that viral infection caused short-term systemic IR. Virally-induced interferon-γ (IFN-γ) directly targeted skeletal muscle to downregulate the insulin receptor but did not cause loss of glycemic control because of a compensatory increase of insulin production. Hyperinsulinemia enhanced antiviral immunity through direct stimulation of CD8+ effector T cell function. In pre-diabetic mice with hepatic IR caused by diet-induced obesity, infection resulted in loss of glycemic control. Thus, upon pathogen encounter, the immune system transiently reduces insulin sensitivity of skeletal muscle to induce hyperinsulinemia and promote antiviral immunity, which derails to glucose intolerance in pre-diabetic obese subjects. VIDEO ABSTRACT.
Asunto(s)
Diabetes Mellitus/inmunología , Resistencia a la Insulina , Insulina/inmunología , Interferón gamma/metabolismo , Músculo Esquelético/metabolismo , Obesidad/inmunología , Virosis/complicaciones , Animales , Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus/etiología , Diabetes Mellitus/metabolismo , Diabetes Mellitus/virología , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Humanos , Hiperinsulinismo , Insulina/sangre , Masculino , Ratones , Obesidad/complicaciones , Obesidad/metabolismo , Obesidad/virología , Receptor de Insulina/genética , Receptor de Insulina/metabolismoRESUMEN
Natural killer (NK) cells are innate lymphoid cells, and their presence within human tumors correlates with better prognosis. However, the mechanisms by which NK cells control tumors in vivo are unclear. Here, we used reflectance confocal microscopy (RCM) imaging in humans and in mice to visualize tumor architecture in vivo. We demonstrated that signaling via the NK cell receptor NKp46 (human) and Ncr1 (mouse) induced interferon-γ (IFN-γ) secretion from intratumoral NK cells. NKp46- and Ncr1-mediated IFN-γ production led to the increased expression of the extracellular matrix protein fibronectin 1 (FN1) in the tumors, which altered primary tumor architecture and resulted in decreased metastases formation. Injection of IFN-γ into tumor-bearing mice or transgenic overexpression of Ncr1 in NK cells in mice resulted in decreased metastasis formation. Thus, we have defined a mechanism of NK cell-mediated control of metastases in vivo that may help develop NK cell-dependent cancer therapies.
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Antígenos Ly/metabolismo , Fibronectinas/metabolismo , Interferón gamma/metabolismo , Células Asesinas Naturales/metabolismo , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismo , Neoplasias/metabolismo , Animales , Western Blotting , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Ratones , Microscopía Confocal , Metástasis de la Neoplasia/genética , Neoplasias/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genéticaRESUMEN
The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include >200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model.
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Muromegalovirus , Animales , Ratones , Humanos , Citomegalovirus/genética , Citomegalovirus/metabolismo , Secuencia de Bases , Proteínas Virales/genética , Proteínas Virales/metabolismo , Sistemas de Lectura AbiertaRESUMEN
The transcriptional repressor Blimp-1 mediates the terminal differentiation of many cell types, including T cells. Here we identified Hobit (Znf683) as a previously unrecognized homolog of Blimp-1 that was specifically expressed in mouse natural killer T cells (NKT cells). Through studies of Hobit-deficient mice, we found that Hobit was essential for the formation of mature thymic NKT cells. In the periphery, Hobit repressed the accumulation of interferon-γ (IFN-γ)-producing NK1.1(lo) NKT cells at steady state. After antigenic stimulation, Hobit repressed IFN-γ expression, whereas after innate stimulation, Hobit induced granzyme B expression. Thus, reminiscent of the function of Blimp-1 in other lymphocytes, Hobit controlled the maintenance of quiescent, fully differentiated NKT cells and regulated their immediate effector functions.
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Diferenciación Celular/genética , Células T Asesinas Naturales/citología , Células T Asesinas Naturales/inmunología , Secuencia de Aminoácidos , Animales , Diferenciación Celular/inmunología , Citometría de Flujo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Filogenia , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genéticaRESUMEN
Bacteria, such as Fusobacterium nucleatum, are present in the tumor microenvironment. However, the immunological consequences of intra-tumoral bacteria remain unclear. Here, we have shown that natural killer (NK) cell killing of various tumors is inhibited in the presence of various F. nucleatum strains. Our data support that this F. nucleatum-mediated inhibition is mediated by human, but not by mouse TIGIT, an inhibitory receptor present on all human NK cells and on various T cells. Using a library of F. nucleatum mutants, we found that the Fap2 protein of F. nucleatum directly interacted with TIGIT, leading to the inhibition of NK cell cytotoxicity. We have further demonstrated that tumor-infiltrating lymphocytes expressed TIGIT and that T cell activities were also inhibited by F. nucleatum via Fap2. Our results identify a bacterium-dependent, tumor-immune evasion mechanism in which tumors exploit the Fap2 protein of F. nucleatum to inhibit immune cell activity via TIGIT.
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Adenocarcinoma/inmunología , Adenocarcinoma/microbiología , Neoplasias del Colon/inmunología , Neoplasias del Colon/microbiología , Fusobacterium nucleatum/inmunología , Receptores Inmunológicos/inmunología , Escape del Tumor/inmunología , Microambiente Tumoral/inmunología , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Línea Celular , Proliferación Celular , Humanos , Células Asesinas Naturales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Unión ProteicaRESUMEN
COVID-19 vaccines prevent severe forms of the disease, but do not warrant complete protection against breakthrough infections. This could be due to suboptimal mucosal immunity at the site of virus entry, given that all currently approved vaccines are administered via the intramuscular route. In this study, we assessed humoral and cellular immune responses in BALB/c mice after intranasal and intramuscular immunization with adenoviral vector ChAdOx1-S expressing full-length Spike protein of SARS-CoV-2. We showed that both routes of vaccination induced a potent IgG antibody response, as well as robust neutralizing capacity, but intranasal vaccination elicited a superior IgA antibody titer in the sera and in the respiratory mucosa. Bronchoalveolar lavage from intranasally immunized mice efficiently neutralized SARS-CoV-2, which has not been the case in intramuscularly immunized group. Moreover, substantially higher percentages of epitope-specific CD8 T cells exhibiting a tissue resident phenotype were found in the lungs of intranasally immunized animals. Finally, both intranasal and intramuscular vaccination with ChAdOx1-S efficiently protected the mice after the challenge with recombinant herpesvirus expressing the Spike protein. Our results demonstrate that intranasal application of adenoviral vector ChAdOx1-S induces superior mucosal immunity and therefore could be a promising strategy for putting the COVID-19 pandemic under control.
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COVID-19 , Vacunas Virales , Adenoviridae/genética , Administración Intranasal , Animales , Anticuerpos Antivirales , COVID-19/prevención & control , Vacunas contra la COVID-19 , Humanos , Inmunidad Celular , Inmunidad Mucosa , Ratones , Ratones Endogámicos BALB C , Pandemias/prevención & control , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Vacunación/métodosRESUMEN
Broad tissue tropism of cytomegaloviruses (CMVs) is facilitated by different glycoprotein entry complexes, which are conserved between human CMV (HCMV) and murine CMV (MCMV). Among the wide array of cell types susceptible to the infection, mononuclear phagocytes (MNPs) play a unique role in the pathogenesis of the infection as they contribute both to the virus spread and immune control. CMVs have dedicated numerous genes for the efficient infection and evasion of macrophages and dendritic cells. In this study, we have characterized the properties and function of M116, a previously poorly described but highly transcribed MCMV gene region that encodes M116.1p, a novel protein necessary for the efficient infection of MNPs and viral spread in vivo. Our study further revealed that M116.1p shares similarities with its positional homologs in HCMV and RCMV, UL116 and R116, respectively, such as late kinetics of expression, N-glycosylation, localization to the virion assembly compartment, and interaction with gH-a member of the CMVs fusion complex. This study, therefore, expands our knowledge about virally encoded glycoproteins that play important roles in viral infectivity and tropism. IMPORTANCE Human cytomegalovirus (HCMV) is a species-specific herpesvirus that causes severe disease in immunocompromised individuals and immunologically immature neonates. Murine cytomegalovirus (MCMV) is biologically similar to HCMV, and it serves as a widely used model for studying the infection, pathogenesis, and immune responses to HCMV. In our previous work, we have identified the M116 ORF as one of the most extensively transcribed regions of the MCMV genome without an assigned function. This study shows that the M116 locus codes for a novel protein, M116.1p, which shares similarities with UL116 and R116 in HCMV and RCMV, respectively, and is required for the efficient infection of mononuclear phagocytes and virus spread in vivo. Furthermore, this study establishes the α-M116 monoclonal antibody and MCMV mutants lacking M116, generated in this work, as valuable tools for studying the role of macrophages and dendritic cells in limiting CMV infection following different MCMV administration routes.
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Sistema Mononuclear Fagocítico/virología , Muromegalovirus/fisiología , Proteínas del Envoltorio Viral/metabolismo , Animales , Fibroblastos/metabolismo , Fibroblastos/virología , Glicosilación , Infecciones por Herpesviridae/virología , Glicoproteínas de Membrana/metabolismo , Ratones , Sistema Mononuclear Fagocítico/metabolismo , Transcripción Genética , Proteínas del Envoltorio Viral/genética , Virión/metabolismo , Ensamble de Virus , Internalización del Virus , Replicación ViralRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.
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Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Dominios Proteicos , Proteínas Recombinantes de Fusión/metabolismo , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Sitios de Unión , Sitios de Unión de Anticuerpos , COVID-19/prevención & control , Chlorocebus aethiops , Femenino , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Inmunoglobulina G/uso terapéutico , Ratones Transgénicos , Pruebas de Neutralización , Unión Proteica , Proteínas Recombinantes de Fusión/uso terapéutico , SARS-CoV-2/efectos de los fármacos , Células VeroRESUMEN
More than 450 immunologists recently met in Cavtat, Croatia to discuss advances in natural killer (NK) cell biology. The meeting highlighted emerging themes in NK cell responses to viruses, NK cell tolerance and potential use of NK cells in the therapy of malignancies.
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Células Asesinas Naturales/inmunología , Receptores de Células Asesinas Naturales/inmunología , Virosis/inmunología , Animales , Antígenos Virales/inmunología , Croacia , Citotoxicidad Inmunológica , Humanos , Tolerancia Inmunológica , Inmunidad Innata , Interleucinas/inmunología , Transducción de Señal , Interleucina-22RESUMEN
Direct type I interferon (IFN) signaling on T cells is necessary for the proper expansion, differentiation, and survival of responding T cells following infection with viruses prominently inducing type I IFN. The reasons for the abortive response of T cells lacking the type I IFN receptor (Ifnar1(-/-)) remain unclear. We report here that Ifnar1(-/-) T cells were highly susceptible to natural killer (NK) cell-mediated killing in a perforin-dependent manner. Depletion of NK cells prior to lymphocytic choriomeningitis virus (LCMV) infection completely restored the early expansion of Ifnar1(-/-) T cells. Ifnar1(-/-) T cells had elevated expression of natural cytotoxicity triggering receptor 1 (NCR1) ligands upon infection, rendering them targets for NCR1 mediated NK cell attack. Thus, direct sensing of type I IFNs by T cells protects them from NK cell killing by regulating the expression of NCR1 ligands, thereby revealing a mechanism by which T cells can evade the potent cytotoxic activity of NK cells.
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Antígenos Ly/inmunología , Citotoxicidad Inmunológica , Interferón Tipo I/inmunología , Células Asesinas Naturales/inmunología , Coriomeningitis Linfocítica/inmunología , Receptor 1 Gatillante de la Citotoxidad Natural/inmunología , Receptor de Interferón alfa y beta/genética , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Inmunidad Innata , Activación de Linfocitos/inmunología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Perforina/biosíntesis , Infecciones por Rhabdoviridae/inmunología , Transducción de Señal/inmunología , Vesiculovirus/genética , Vesiculovirus/inmunología , Replicación Viral/inmunologíaRESUMEN
The memory CD8 T-cell pool must select for clones that bind immunodominant epitopes with high affinity to efficiently counter reinfection. At the same time, it must retain a level of clonal diversity to allow recognition of pathogens with mutated epitopes. How the level of diversity within the memory pool is controlled is unclear, especially in the context of a selective drive for antigen affinity. We find that preservation of clones that bind the activating antigen with low affinity depends on expression of the transcription factor Eomes in the first days after antigen encounter. Eomes is induced at low activating signal strength and directly drives transcription of the prosurvival protein Bcl-2. At higher signal intensity, T-bet is induced which suppresses Bcl-2 and causes a relative survival advantage for cells of low affinity. Clones activated with high-affinity antigen form memory largely independent of Eomes and have a proliferative advantage over clones that bind the same antigen with low affinity. This causes high-affinity clones to prevail in the memory pool, despite their relative survival deficit. Genetic or therapeutic targeting of the Eomes/Bcl-2 axis reduces the clonal diversity of the memory pool, which diminishes its ability to respond to pathogens carrying mutations in immunodominant epitopes. Thus, we demonstrate on a molecular level how sufficient diversity of the memory pool is established in an environment of affinity-based selection.
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Apoptosis/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Proteínas de Dominio T Box/inmunología , Animales , Variación Antigénica/inmunología , Supervivencia Celular/inmunología , Células Cultivadas , Selección Clonal Mediada por Antígenos/genética , Selección Clonal Mediada por Antígenos/inmunología , Regulación de la Expresión Génica/inmunología , Activación de Linfocitos , Ratones , Células Precursoras de Linfocitos T/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal , Proteínas de Dominio T Box/genéticaRESUMEN
Interleukin-1 receptor 8 (IL-1R8, also known as single immunoglobulin IL-1R-related receptor, SIGIRR, or TIR8) is a member of the IL-1 receptor (ILR) family with distinct structural and functional characteristics, acting as a negative regulator of ILR and Toll-like receptor (TLR) downstream signalling pathways and inflammation. Natural killer (NK) cells are innate lymphoid cells which mediate resistance against pathogens and contribute to the activation and orientation of adaptive immune responses. NK cells mediate resistance against haematopoietic neoplasms but are generally considered to play a minor role in solid tumour carcinogenesis. Here we report that IL-1R8 serves as a checkpoint for NK cell maturation and effector function. Its genetic blockade unleashes NK-cell-mediated resistance to hepatic carcinogenesis, haematogenous liver and lung metastasis, and cytomegalovirus infection.
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Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Neoplasias Hepáticas/inmunología , Muromegalovirus/inmunología , Receptores de Interleucina-1/inmunología , Animales , Diferenciación Celular/genética , Femenino , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/inmunología , Humanos , Células Asesinas Naturales/metabolismo , Neoplasias Hepáticas/genética , Activación de Linfocitos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/inmunología , Receptores de Interleucina-1/genéticaRESUMEN
Viral immune evasion is currently understood to focus on deflecting CD8 T cell recognition of infected cells by disrupting antigen presentation pathways. We evaluated viral interference with the ultimate step in cytotoxic T cell function, the death of infected cells. The viral inhibitor of caspase-8 activation (vICA) conserved in human cytomegalovirus (HCMV) and murine CMV (MCMV) prevents the activation of caspase-8 and proapoptotic signaling. We demonstrate the key role of vICA from either virus, in deflecting antigen-specific CD8 T cell-killing of infected cells. vICA-deficient mutants, lacking either UL36 or M36, exhibit greater susceptibility to CD8 T cell control than mutants lacking the set of immunoevasins known to disrupt antigen presentation via MHC class I. This difference is evident during infection in the natural mouse host infected with MCMV, in settings where virus-specific CD8 T cells are adoptively transferred. Finally, we identify the molecular mechanism through which vICA acts, demonstrating the central contribution of caspase-8 signaling at a point of convergence of death receptor-induced apoptosis and perforin/granzyme-dependent cytotoxicity.
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Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Interacciones Microbiota-Huesped/inmunología , Evasión Inmune , Linfocitos T Citotóxicos/inmunología , Animales , Apoptosis/inmunología , Caspasa 8/genética , Caspasa 8/metabolismo , Línea Celular , Técnicas de Cocultivo , Citomegalovirus/patogenicidad , Infecciones por Citomegalovirus/virología , Modelos Animales de Enfermedad , Fibroblastos , Granzimas/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Ratones , Ratones Noqueados , Muromegalovirus/genética , Muromegalovirus/inmunología , Muromegalovirus/metabolismo , Mutagénesis , Perforina/genética , Perforina/metabolismo , Receptores de Muerte Celular/metabolismo , Transducción de Señal/inmunología , Linfocitos T Citotóxicos/metabolismo , Imagen de Lapso de Tiempo , Proteínas Virales/genética , Proteínas Virales/inmunología , Proteínas Virales/metabolismoRESUMEN
Human cytomegalovirus is responsible for morbidity and mortality in immune compromised patients and is the leading viral cause of congenital infection. Virus-encoded microRNAs (miRNAs) represent interesting targets for novel antiviral agents. While many cellular targets that augment productive infection have been identified in recent years, regulation of viral genes such as the major viral immediate early protein 72 (IE72) by hcmv-miR-UL112-1 may contribute to both the establishment and the maintenance of latent infection. We employed photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking (PAR-iCLIP) to identify murine cytomegalovirus (MCMV) miRNA targets during lytic infection. While the PAR-iCLIP data were of insufficient quality to obtain a comprehensive list of cellular and viral miRNA targets, the most prominent PAR-iCLIP peak in the MCMV genome mapped to the 3' untranslated region of the major viral immediate early 3 (ie3) transcript. We show that this results from two closely positioned binding sites for the abundant MCMV miRNAs miR-M23-2-3p and miR-m01-2-3p. Their pre-expression significantly impaired viral plaque formation. However, mutation of the respective binding sites did not alter viral fitness during acute or subacute infection in vivo. Furthermore, no differences in the induction of virus-specific CD8+ T cells were observed. Future studies will probably need to go beyond studying immunocompetent laboratory mice housed in pathogen-free conditions to reveal the functional relevance of viral miRNA-mediated regulation of key viral immediate early genes.
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MicroARNs , Muromegalovirus , Humanos , Ratones , Animales , Muromegalovirus/genética , Genes Inmediatos-Precoces , Linfocitos T CD8-positivos/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Citomegalovirus/genética , Regiones no Traducidas 3'RESUMEN
The cloning of herpesviruses as bacterial artificial chromosomes (BACs) has revolutionized the study of herpesvirus biology, allowing rapid and precise manipulation of viral genomes. Several clinical strains of human cytomegalovirus (HCMV) have been cloned as BACs; however, no low-passage strains of murine CMV (MCMV), which provide a model mimicking these isolates, have been cloned. Here, the low-passage G4 strain of was BAC cloned. G4 carries an m157 gene that does not ligate the natural killer (NK) cell-activating receptor, Ly49H, meaning that unlike laboratory strains of MCMV, this virus replicates well in C57BL/6 mice. This BAC clone exhibited normal replication during acute infection in the spleen and liver but was attenuated for salivary gland tropism. Next-generation sequencing revealed a C-to-A mutation at nucleotide position 188422, located in the 3' untranslated region of sgg1, a spliced gene critical for salivary gland tropism. Repair of this mutation restored tropism for the salivary glands. Transcriptional analysis revealed a novel spliced gene within the sgg1 locus. This small open reading frame (ORF), sgg1.1, starts at the 3' end of the first exon of sgg1 and extends exon 2 of sgg1. This shorter spliced gene is prematurely terminated by the nonsense mutation at nt 188422. Sequence analysis of tissue culture-passaged virus demonstrated that sgg1.1 was stable, although other mutational hot spots were identified. The G4 BAC will allow in vivo studies in a broader range of mice, avoiding the strong NK cell responses seen in B6 mice with other MCMV BAC-derived MCMVs.IMPORTANCE Murine cytomegalovirus (MCMV) is widely used as a model of human CMV (HCMV) infection. However, this model relies on strains of MCMV that have been serially passaged in the laboratory for over four decades. These laboratory strains have been cloned as bacterial artificial chromosomes (BACs), which permits rapid and precise manipulation. Low-passage strains of MCMV add to the utility of the mouse model of HCMV infection but do not exist as cloned BACs. This study describes the first such low-passage MCMV BAC. This BAC-derived G4 was initially attenuated in vivo, with subsequent full genomic sequencing revealing a novel spliced transcript required for salivary gland tropism. These data suggest that MCMV, like HCMV, undergoes tissue culture adaptation that can limit in vivo growth and supports the use of BAC clones as a way of standardizing viral strains and minimizing interlaboratory strain variation.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Muromegalovirus/genética , Glándulas Salivales/virología , Tropismo/fisiología , Animales , ADN Recombinante , Femenino , Genoma Viral , Infecciones por Herpesviridae/virología , Humanos , Células Asesinas Naturales , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mutación , Sistemas de Lectura Abierta , Proteínas Virales/genéticaRESUMEN
To ensure productive infection, herpesviruses utilize tegument proteins and nonstructural regulatory proteins to counteract cellular defense mechanisms and to reprogram cellular pathways. The M25 proteins of mouse cytomegalovirus (MCMV) belong to the betaherpesvirus UL25 gene family that encodes viral proteins implicated with regulatory functions. Through affinity purification and mass spectrometric analysis, we discovered the tumor suppressor protein p53 as a host factor interacting with the M25 proteins. M25-p53 interaction in infected and transfected cells was confirmed by coimmunoprecipitation. Moreover, the proteins colocalized in nuclear dot-like structures upon both infection and inducible expression of the two M25 isoforms. p53 accumulated in wild-type MCMV-infected cells, while this did not occur upon infection with a mutant lacking the M25 gene. Both M25 proteins were able to mediate the effect, identifying them as the first CMV proteins responsible for p53 accumulation during infection. Interaction with M25 proteins led to substantial prolongation of the half-life of p53. In contrast to the higher abundance of the p53 protein in wild-type MCMV-infected cells, the transcript levels of the prominent p53 target genes Cdkn1a and Mdm2 were diminished compared to cells infected with the ΔM25 mutant, and this was associated with reduced binding of p53 to responsive elements within the respective promoters. Notably, the productivity of the M25 deletion mutant was partially rescued on p53-negative fibroblasts. We propose that the MCMV M25 proteins sequester p53 molecules in the nucleus of infected cells, reducing their availability for activating a subset of p53-regulated genes, thereby dampening the antiviral role of p53.IMPORTANCE Host cells use a number of factors to defend against viral infection. Viruses are, however, in an arms race with their host cells to overcome these defense mechanisms. The tumor suppressor protein p53 is an important sensor of cell stress induced by oncogenic insults or viral infections, which upon activation induces various pathways to ensure the integrity of cells. Viruses have to counteract many functions of p53, but complex DNA viruses such as cytomegaloviruses may also utilize some p53 functions for their own benefit. In this study, we discovered that the M25 proteins of mouse cytomegalovirus interact with p53 and mediate its accumulation during infection. Interaction with the M25 proteins sequesters p53 molecules in nuclear dot-like structures, limiting their availability for activation of a subset of p53-regulated target genes. Understanding the interaction between viral proteins and p53 may allow to develop new therapeutic strategies against cytomegalovirus and other viruses.