RESUMEN
Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.
Asunto(s)
Transporte Activo de Núcleo Celular , Cápside/metabolismo , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestructura , Vesículas Transportadoras/ultraestructura , Animales , Cápside/ultraestructura , Chlorocebus aethiops , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Herpesvirus Humano 1/metabolismo , Herpesvirus Suido 1/metabolismo , Membrana Nuclear/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Dímeros de Pirimidina , Dispersión del Ángulo Pequeño , Vesículas Transportadoras/metabolismo , Células Vero , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Tuberculosis remains a global health problem so that a more effective vaccine than bacillus Calmette-Guérin is urgently needed. Cytomegaloviruses persist lifelong in vivo and induce powerful immune and increasing ("inflationary") responses, making them attractive vaccine vectors. We have used an m1-m16-deleted recombinant murine CMV (MCMV) expressing Mycobacterium tuberculosis Ag 85A to show that infection of mice with this recombinant significantly reduces the mycobacterial load after challenge with M. tuberculosis, whereas control empty virus has a lesser effect. Both viruses induce immune responses to H-2(d)-restricted epitopes of MCMV pp89 and M18 Ags characteristic of infection with other MCMVs. A low frequency of 85A-specific memory cells could be revealed by in vivo or in vitro boosting or after challenge with M. tuberculosis. Kinetic analysis of M. tuberculosis growth in the lungs of CMV-infected mice shows early inhibition of M. tuberculosis growth abolished by treatment with NK-depleting anti-asialo ganglio-N-tetraosylceramide Ab. Microarray analysis of the lungs of naive and CMV-infected mice shows increased IL-21 mRNA in infected mice, whereas in vitro NK assays indicate increased levels of NK activity. These data indicate that activation of NK cells by MCMV provides early nonspecific protection against M. tuberculosis, potentiated by a weak 85A-specific T cell response, and they reinforce the view that the innate immune system plays an important role in both natural and vaccine-induced protection against M. tuberculosis.
Asunto(s)
Epítopos/inmunología , Vectores Genéticos , Muromegalovirus , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Pulmonar/prevención & control , Animales , Epítopos/genética , Femenino , Antígeno de Histocompatibilidad H-2D/genética , Antígeno de Histocompatibilidad H-2D/inmunología , Interleucinas/genética , Interleucinas/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/genética , Vacunas contra la Tuberculosis/genética , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/patologíaRESUMEN
Human cytomegalovirus (HCMV) forms two gH/gL glycoprotein complexes, gH/gL/gO and gH/gL/pUL(128,130,131A), which determine the tropism, the entry pathways and the mode of spread of the virus. For murine cytomegalovirus (MCMV), which serves as a model for HCMV, a gH/gL/gO complex functionally homologous to the HCMV gH/gL/gO complex has been described. Knock-out of MCMV gO does impair, but not abolish, virus spread indicating that also MCMV might form an alternative gH/gL complex. Here, we show that the MCMV CC chemokine MCK-2 forms a complex with the glycoprotein gH, a complex which is incorporated into the virion. We could additionally show that mutants lacking both, gO and MCK-2 are not able to produce infectious virus. Trans-complementation of these double mutants with either gO or MCK-2 showed that both proteins can promote infection of host cells, although through different entry pathways. MCK-2 has been extensively studied in vivo by others. It has been shown to be involved in attracting cells for virus dissemination and in regulating antiviral host responses. We now show that MCK-2, by forming a complex with gH, strongly promotes infection of macrophages in vitro and in vivo. Thus, MCK-2 may play a dual role in MCMV infection, as a chemokine regulating the host response and attracting specific target cells and as part of a glycoprotein complex promoting entry into cells crucial for virus dissemination.
Asunto(s)
Quimiocinas CC/metabolismo , Infecciones por Herpesviridae/inmunología , Inmunidad Innata , Macrófagos/inmunología , Muromegalovirus/fisiología , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/metabolismo , Internalización del Virus , Animales , Línea Celular , Células Cultivadas , Quimiocinas CC/química , Quimiocinas CC/genética , Femenino , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Hígado/inmunología , Hígado/patología , Hígado/virología , Macrófagos/patología , Macrófagos/virología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Macrófagos Peritoneales/virología , Ratones , Ratones Endogámicos BALB C , Muromegalovirus/inmunología , Mutación , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Organismos Libres de Patógenos Específicos , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas Virales/química , Proteínas Virales/genética , Virión/inmunología , Virión/fisiologíaRESUMEN
Human gammaherpesviruses cause morbidity and mortality associated with infection and transformation of lymphoid and endothelial cells. Knowledge of cell types involved in virus dissemination from primary virus entry to virus latency is fundamental for the understanding of gammaherpesvirus pathogenesis. However, the inability to directly trace cell types with respect to virus dissemination pathways has prevented definitive conclusions regarding the relative contribution of individual cell types. Here, we describe that the route of infection affects gammaherpesvirus dissemination pathways. We constructed a recombinant murine gammaherpesvirus 68 (MHV-68) variant harboring a cassette which switches fluorescent markers in a Cre-dependent manner. Since the recombinant virus which was constructed on the wild-type background was attenuated, in this study we used an M1-deleted version, which infected mice with normal kinetics. Infection of Cre-transgenic mice with this convertible virus was used to estimate the quantitative contribution of defined cell types to virus productivity and dissemination during the acute phase of MHV-68 infection. In systemic infection, we found splenic vascular endothelial cells (EC) among the first and main cells to produce virus. After local infection, the contribution of EC to splenic virus production did not represent such early kinetics. However, at later time points, B cell-derived viruses dominated splenic productivity independently of systemic or local infection. Systemic versus local infection also governed the cell types involved in loading peritoneal exudate cells, leading to latency in F4/80- and CD11b-positive target cells. Systemic infection supported EC-driven dissemination, whereas local infection supported B cell-driven dissemination.
Asunto(s)
Infecciones por Herpesviridae/virología , Rhadinovirus/patogenicidad , Infecciones Tumorales por Virus/virología , Tropismo Viral , Replicación Viral , Animales , Linfocitos B/virología , Línea Celular , Células Endoteliales/virología , Genes Reporteros , Infecciones por Herpesviridae/patología , Estudios Longitudinales , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Rhadinovirus/genética , Rhadinovirus/crecimiento & desarrollo , Rhadinovirus/fisiología , Bazo/virología , Coloración y Etiquetado/métodos , Infecciones Tumorales por Virus/patologíaRESUMEN
The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC(-/-)), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system.
Asunto(s)
Inmunidad Adaptativa , Inmunodeficiencia Variable Común/inmunología , Infecciones por Herpesviridae/inmunología , Macrófagos/inmunología , Muromegalovirus/inmunología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Línea Celular , Inmunodeficiencia Variable Común/genética , Inmunodeficiencia Variable Común/patología , Inmunodeficiencia Variable Común/virología , Inhibidores de Cisteína Proteinasa/farmacología , Fibroblastos/inmunología , Fibroblastos/patología , Fibroblastos/virología , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/patología , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Macrófagos/patología , Macrófagos/virología , Ratones , Ratones Noqueados , Muromegalovirus/genética , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed.
Asunto(s)
Infecciones por Citomegalovirus/genética , Citomegalovirus/genética , Regulación Viral de la Expresión Génica/genética , Muromegalovirus/genética , Animales , Western Blotting , Genes Reporteros , Hibridación Fluorescente in Situ , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Células 3T3 NIH , Replicón/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cytomegaloviruses express large amounts of viral miRNAs during lytic infection, yet, they only modestly alter the cellular miRNA profile. The most prominent alteration upon lytic murine cytomegalovirus (MCMV) infection is the rapid degradation of the cellular miR-27a and miR-27b. Here, we report that this regulation is mediated by the â¼1.7 kb spliced and highly abundant MCMV m169 transcript. Specificity to miR-27a/b is mediated by a single, apparently optimized, miRNA binding site located in its 3'-UTR. This site is easily and efficiently retargeted to other cellular and viral miRNAs by target site replacement. Expression of the 3'-UTR of m169 by an adenoviral vector was sufficient to mediate its function, indicating that no other viral factors are essential in this process. Degradation of miR-27a/b was found to be accompanied by 3'-tailing and -trimming. Despite its dramatic effect on miRNA stability, we found this interaction to be mutual, indicating potential regulation of m169 by miR-27a/b. Most interestingly, three mutant viruses no longer able to target miR-27a/b, either due to miRNA target site disruption or target site replacement, showed significant attenuation in multiple organs as early as 4 days post infection, indicating that degradation of miR-27a/b is important for efficient MCMV replication in vivo.
Asunto(s)
Regiones no Traducidas 3'/genética , Infecciones por Citomegalovirus/virología , MicroARNs/metabolismo , Muromegalovirus/fisiología , ARN Viral/metabolismo , Replicación Viral/genética , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sitios de Unión , Línea Celular , Regulación hacia Abajo/genética , Regulación de la Expresión Génica/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , MicroARNs/genética , Muromegalovirus/genética , Mutación , Procesamiento Postranscripcional del ARN , Estabilidad del ARN/genética , ARN Viral/genética , Análisis de Secuencia de ARNRESUMEN
The herpesvirus replication cycle comprises maturation processes in the nucleus and cytoplasm of the infected cells. After their nuclear assembly viral capsids translocate via primary envelopment towards the cytoplasm. This event is mediated by the nuclear envelopment complex, which is composed by two conserved viral proteins belonging to the UL34 and UL31 protein families. Here, we generated recombinant viruses, which express affinity-tagged pM50 and/or pM53, the pUL34 and pUL31 homologues of the murine cytomegalovirus. We extracted pM50- and pM53-associated protein complexes from infected cells and analysed their composition after affinity purification by mass spectrometry. We observed reported interaction partners and identified new putative protein-protein interactions for both proteins. Endophilin-A2 was observed as the most prominent cellular partner of pM50. We found that endophilin-A2 binds to pM50 directly, and this interaction seems to be conserved in the pUL34 family.
Asunto(s)
Aciltransferasas/metabolismo , Muromegalovirus/fisiología , Proteínas Mutantes Quiméricas/metabolismo , Proteínas Virales/metabolismo , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/genética , Animales , Citosol/metabolismo , Citosol/virología , Expresión Génica , Interacciones Huésped-Patógeno , Espectrometría de Masas , Ratones , Proteínas Mutantes Quiméricas/genética , Membrana Nuclear/metabolismo , Membrana Nuclear/virología , Unión Proteica , Mapeo de Interacción de Proteínas , ARN Interferente Pequeño/genética , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genética , Liberación del VirusRESUMEN
Dominant-negative (DN) mutants are powerful tools for studying essential protein-protein interactions. A systematic genetic screen of the essential murine cytomegalovirus (MCMV) protein pM53 identified the accumulation of inhibitory mutations within conserved region 2 (CR2) and CR4. The strong inhibitory potential of these CR4 mutants is characterized by a particular phenotype. The DN effect of the small insertion mutations in CR2 was too weak to analyze (M. Popa, Z. Ruzsics, M. Lötzerich, L. Dölken, C. Buser, P. Walther, and U. H. Koszinowski, J. Virol. 84:9035-9046, 2010); therefore, the present study describes the construction of M53 alleles lacking CR2 (either completely or partially) and subsequent examination of the DN effect on MCMV replication upon conditional expression. Overexpression of CR2-deficient pM53 inhibited virus production by about 10,000-fold. This was due to interference with capsid export from the nucleus and viral genome cleavage/packaging. In addition, the fate of the nuclear envelopment complex in the presence of DN pM53 overexpression was analyzed. The CR2 mutants were able to bind to pM50, albeit to a lesser extent than the wild-type protein, and relocalized the wild-type nuclear envelope complex in infected cells. Unlike the CR4 DN, the CR2 DN mutants did not affect the stability of pM50.
Asunto(s)
Proteínas de la Cápside/genética , Muromegalovirus/genética , Membrana Nuclear/virología , Proteínas Nucleares/genética , Replicación Viral/genética , Alelos , Animales , Southern Blotting , Western Blotting , Proteínas de la Cápside/metabolismo , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Prueba de Complementación Genética , Inmunoprecipitación , Ratones , Microscopía Confocal , Microscopía Electrónica de Transmisión , Muromegalovirus/crecimiento & desarrollo , Mutación/genética , Proteínas Nucleares/metabolismo , Plásmidos/genética , Reacción en Cadena de la PolimerasaRESUMEN
Human cytomegalovirus (HCMV) can infect many different cell types in vivo. Two gH/gL complexes are used for entry into cells. gH/gL/pUL(128,130,131A) shows no selectivity for its host cell, whereas formation of a gH/gL/gO complex only restricts the tropism mainly to fibroblasts. Here, we describe that depending on the cell type in which virus replication takes place, virus carrying the gH/gL/pUL(128,130,131A) complex is either released or retained cell-associated. We observed that virus spread in fibroblast cultures was predominantly supernatant-driven, whereas spread in endothelial cell (EC) cultures was predominantly focal. This was due to properties of virus released from fibroblasts and EC. Fibroblasts released virus which could infect both fibroblasts and EC. In contrast, EC released virus which readily infected fibroblasts, but was barely able to infect EC. The EC infection capacities of virus released from fibroblasts or EC correlated with respectively high or low amounts of gH/gL/pUL(128,130,131A) in virus particles. Moreover, we found that focal spread in EC cultures could be attributed to EC-tropic virus tightly associated with EC and not released into the supernatant. Preincubation of fibroblast-derived virus progeny with EC or beads coated with pUL131A-specific antibodies depleted the fraction that could infect EC, and left a fraction that could predominantly infect fibroblasts. These data strongly suggest that HCMV progeny is composed of distinct virus populations. EC specifically retain the EC-tropic population, whereas fibroblasts release EC-tropic and non EC-tropic virus. Our findings offer completely new views on how HCMV spread may be controlled by its host cells.
Asunto(s)
Infecciones por Citomegalovirus/virología , Citomegalovirus/fisiología , Endotelio Vascular/virología , Fibroblastos/virología , Tropismo Viral/fisiología , Secuencia de Bases , Células Cultivadas , Citomegalovirus/patogenicidad , Endotelio Vascular/metabolismo , Fibroblastos/metabolismo , Prepucio/citología , Interacciones Huésped-Patógeno , Humanos , Masculino , Datos de Secuencia Molecular , Proteínas Virales/metabolismo , Internalización del Virus , Replicación Viral/fisiologíaRESUMEN
Cytomegalovirus (CMV) is frequently transmitted by solid organ transplantation and is associated with graft failure. By forming the boundary between circulation and organ parenchyma, endothelial cells (EC) are suited for bidirectional virus spread from and to the transplant. We applied Cre/loxP-mediated green-fluorescence-tagging of EC-derived murine CMV (MCMV) to quantify the role of infected EC in transplantation-associated CMV dissemination in the mouse model. Both EC- and non-EC-derived virus originating from infected Tie2-cre(+) heart and kidney transplants were readily transmitted to MCMV-naïve recipients by primary viremia. In contrast, when a Tie2-cre(+) transplant was infected by primary viremia in an infected recipient, the recombined EC-derived virus poorly spread to recipient tissues. Similarly, in reverse direction, EC-derived virus from infected Tie2-cre(+) recipient tissues poorly spread to the transplant. These data contradict any privileged role of EC in CMV dissemination and challenge an indiscriminate applicability of the primary and secondary viremia concept of virus dissemination.
Asunto(s)
Infecciones por Citomegalovirus/virología , Células Endoteliales/virología , Muromegalovirus/patogenicidad , Animales , Endotelio Vascular/virología , Corazón/virología , Trasplante de Corazón/efectos adversos , Riñón/virología , Trasplante de Riñón/efectos adversos , Ratones , Ratones Transgénicos , Viremia/virologíaRESUMEN
Members of the alpha- and beta-subfamily of herpesviridae encode glycoproteins that specifically bind to the Fc part of immunoglobulin (Ig)G. Plasma membrane resident herpesviral Fc receptors seem to prevent virus-specific IgG from activating antibody-dependent effector functions. We show that the mouse cytomegalovirus (MCMV) molecule fcr-1 promotes a rapid down-regulation of NKG2D ligands murine UL16-binding protein like transcript (MULT)-1 and H60 from the cell surface. Deletion of the m138/fcr-1 gene from the MCMV genome attenuates viral replication to natural killer (NK) cell response in an NKG2D-dependent manner in vivo. A distinct N-terminal module within the fcr-1 ectodomain in conjunction with the fcr-1 transmembrane domain was required to dispose MULT-1 to degradation in lysosomes. In contrast, down-modulation of H60 required the complete fcr-1 ectodomain, implying independent modes of fcr-1 interaction with the NKG2D ligands. The results establish a novel viral strategy for down-modulating NK cell responses and highlight the impressive diversity of Fc receptor functions.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación hacia Abajo , Antígenos de Histocompatibilidad Clase I/metabolismo , Glicoproteínas de Membrana/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Muromegalovirus/metabolismo , Receptores Fc/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas Virales/metabolismo , Animales , Proteínas Portadoras/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Inmunoglobulinas/inmunología , Ligandos , Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Antígenos de Histocompatibilidad Menor/inmunología , Muromegalovirus/fisiología , Células 3T3 NIH , Subfamilia K de Receptores Similares a Lectina de Células NK , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Receptores Inmunológicos/inmunología , Receptores de Células Asesinas Naturales , Replicación Viral/fisiologíaRESUMEN
Murine cytomegalovirus (MCMV) Smith strain has been cloned as a bacterial artificial chromosome (BAC) named pSM3fr and used for analysis of virus gene functions in vitro and in vivo. When sequencing the complete BAC genome, we identified a frameshift mutation within the open reading frame (ORF) encoding MCMV chemokine homologue MCK-2. This mutation would result in a truncated MCK-2 protein. When mice were infected with pSM3fr-derived virus, we observed reduced virus production in salivary glands, which could be reverted by repair of the frameshift mutation. When looking for the source of the mutation, we consistently found that virus stocks of cell culture-passaged MCMV Smith strain are mixtures of viruses with or without the MCK-2 mutation. We conclude that the MCK-2 mutation in the pSM3fr BAC is the result of clonal selection during the BAC cloning procedure.
Asunto(s)
Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Cromosomas Artificiales Bacterianos , Mutación del Sistema de Lectura , Muromegalovirus/genética , Muromegalovirus/patogenicidad , Glándulas Salivales/virología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Eliminación de Secuencia , Carga Viral , VirulenciaRESUMEN
Major histocompatibility complex class I (MHC I) molecules present antigenic peptides for CD8(+) T-cell recognition. Prior to cell surface expression, proper MHC I loading is conducted by the peptide-loading complex (PLC), composed of the MHC I heavy chain (HC) and ß(2)-microglobulin (ß(2)m), the peptide transporter TAP, and several chaperones, including tapasin. Tapasin connects peptide-receptive MHC I molecules to the PLC, thereby facilitating loading of high-affinity peptides onto MHC I. To cope with CD8(+) T-cell responses, human cytomegalovirus (HCMV) encodes several posttranslational strategies inhibiting peptide transport and MHC I biogenesis which have been studied extensively in transfected cells. Here we analyzed assembly of the PLC in naturally HCMV-infected fibroblasts throughout the protracted replication cycle. MHC I incorporation into the PLC was absent early in HCMV infection. Subsequently, tapasin neosynthesis became strongly reduced, while tapasin steady-state levels diminished only slowly in infected cells, revealing a blocked synthesis rather than degradation. Tapasin mRNA levels were continuously downregulated during infection, while tapasin transcripts remained stable and long-lived. Taking advantage of a novel method by which de novo transcribed RNA is selectively labeled and analyzed, an immediate decline of tapasin transcription was seen, followed by downregulation of TAP2 and TAP1 gene expression. However, upon forced expression of tapasin in HCMV-infected cells, repair of MHC I incorporation into the PLC was relatively inefficient, suggesting an additional level of HCMV interference. The data presented here document a two-pronged coordinated attack on tapasin function by HCMV.
Asunto(s)
Presentación de Antígeno , Citomegalovirus/patogenicidad , Antígenos de Histocompatibilidad Clase I/inmunología , Evasión Inmune , Proteínas de Transporte de Membrana/biosíntesis , Transcripción Genética , Células Cultivadas , Citomegalovirus/inmunología , Fibroblastos/virología , HumanosRESUMEN
The gene M94 of murine cytomegalovirus (MCMV) as well as its homologues UL16 in alphaherpesviruses is involved in viral morphogenesis. For a better understanding of its role in the viral life cycle, a library of random M94 mutants was generated by modified transposon-based linker scanning mutagenesis. A comprehensive set of M94 mutants was reinserted into the MCMV genome and tested for their capacity to complement the M94 null mutant. Thereby, 34 loss-of-function mutants of M94 were identified, which were tested in a second screen for their capacity to inhibit virus replication. This analysis identified two N-terminal insertion mutants of M94 with a dominant negative effect. We compared phenotypes induced by the conditional expression of these dominant negative M94 alleles with the null phenotype of the M94 deletion. The viral gene expression cascade and the nuclear morphogenesis steps were not affected in either setting. In both cases, however, secondary envelopment did not proceed in the absence of functional M94, and capsids subsequently accumulated in the center of the cytoplasmic assembly complex. In addition, deletion of M94 resulted in a block of cell-to-cell spread. Moreover, the dominant negative mutant of M94 demonstrated a defect in interacting with M99, the UL11 homologue of MCMV.
Asunto(s)
Muromegalovirus/fisiología , Proteínas Virales/metabolismo , Ensamble de Virus , ADN Viral/química , ADN Viral/genética , Eliminación de Gen , Prueba de Complementación Genética , Datos de Secuencia Molecular , Muromegalovirus/genética , Mutagénesis , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Análisis de Secuencia de ADN , Proteínas Virales/genéticaRESUMEN
Micro (mi)RNAs are small non-coding RNAs that regulate the expression of their targets' messenger RNAs through both translational inhibition and regulation of target RNA stability. Recently, a number of viruses, particularly of the herpesvirus family, have been shown to express their own miRNAs to control both viral and cellular transcripts. Although some targets of viral miRNAs are known, their function in a physiologically relevant infection remains to be elucidated. As such, no in vivo phenotype of a viral miRNA knock-out mutant has been described so far. Here, we report on the first functional phenotype of a miRNA knock-out virus in vivo. During subacute infection of a mutant mouse cytomegalovirus lacking two viral miRNAs, virus production is selectively reduced in salivary glands, an organ essential for virus persistence and horizontal transmission. This phenotype depends on several parameters including viral load and mouse genetic background, and is abolished by combined but not single depletion of natural killer (NK) and CD4+ T cells. Together, our results point towards a miRNA-based immunoevasion mechanism important for long-term virus persistence.
Asunto(s)
Infecciones por Citomegalovirus/genética , Citomegalovirus/genética , Citomegalovirus/patogenicidad , MicroARNs/fisiología , Glándulas Salivales/virología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Citomegalovirus/inmunología , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/virología , Regulación Viral de la Expresión Génica/fisiología , Técnicas de Inactivación de Genes , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , MicroARNs/genética , Modelos Biológicos , Organismos Modificados Genéticamente , ARN Viral/genética , ARN Viral/fisiología , Glándulas Salivales/metabolismo , Glándulas Salivales/patología , Vacunas Atenuadas/genética , Carga Viral/genéticaRESUMEN
BACKGROUND: The polyomaviruses WUPyV and KIPyV have been detected in various sample types including feces indicating pathogenicity in the gastrointestinal (GI) system. However, quantitative viral load data from other simultaneously collected sample types are missing. As a consequence, primary replication in the GI system cannot be differentiated from swallowed virus from the respiratory tract. Here we present a retrospective quantitative longitudinal analysis in simultaneously harvested specimens from different organ sites of patients undergoing hematopoietic stem cell transplantation (HSCT). This allows the definition of sample types where deoxyribonucleic acid (DNA) detection can be expected and, as a consequence, the identification of their primary replication site. FINDINGS: Viral DNA loads from 37 patients undergoing HSCT were quantified in respiratory tract secretions (RTS), stool and urine samples as well as in leukocytes (n = 449). Leukocyte-associated virus could not be found. WUPyV was found in feces, RTS and urine samples of an infant, while KIPyV was repeatedly detected in RTS and stool samples of 4 adult patients.RTS and stool samples were matched to determine the viral load difference showing a mean difference of 2.3 log copies/ml (p < 0.001). CONCLUSIONS: The data collected in this study suggest that virus detection in the GI tract results from swallowed virus from the respiratory tract (RT). We conclude that shedding from the RT should be ruled out before viral DNA detection in the feces can be correlated to GI symptoms.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/efectos adversos , Infecciones por Polyomavirus/virología , Poliomavirus/clasificación , Poliomavirus/aislamiento & purificación , Adulto , Heces/virología , Femenino , Enfermedades Gastrointestinales/virología , Humanos , Lactante , Estudios Longitudinales , Masculino , Infecciones del Sistema Respiratorio/virología , Estudios Retrospectivos , Esputo/virología , Orina/virologíaRESUMEN
Human tumors frequently express membrane-bound or soluble NK group 2, member D (NKG2D) ligands. This results in chronic engagement of NKG2D on the surfaces of NK and CD8(+) T cells and rapid internalization of the receptor. Although it is well appreciated that this phenomenon impairs NKG2D-dependent function, careful analysis of NKG2D-independent functions in cells chronically stimulated through NKG2D is lacking. Using a mouse model of chronic NKG2D ligand expression, we show that constant exposure to NKG2D ligands does not functionally impair NK cells and CD8(+) T cells in the context of viral infection.
Asunto(s)
Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/fisiología , Proteínas Asociadas a Matriz Nuclear/fisiología , Proteínas de Transporte Nucleocitoplasmático/fisiología , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Diferenciación Celular/inmunología , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad , Células Asesinas Naturales/citología , Células Asesinas Naturales/virología , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Muromegalovirus/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/antagonistas & inhibidores , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Factores de TiempoRESUMEN
A mouse cytomegalovirus (MCMV) gene conferring interferon (IFN) resistance was identified. This gene, M27, encodes a 79-kD protein that selectively binds and down-regulates for signal transducer and activator of transcription (STAT)-2, but it has no effect on STAT1 activation and signaling. The absence of pM27 conferred MCMV susceptibility to type I IFNs (alpha/beta), but it had a much more dramatic effect on type II IFNs (gamma) in vitro and in vivo. A comparative analysis of M27(+) and M27(-) MCMV revealed that the antiviral efficiency of IFN-gamma was partially dependent on the synergistic action of type I IFNs that required STAT2. Moreover, STAT2 was directly activated by IFN-gamma. This effect required IFN receptor expression and was independent of type I IFNs. IFN-gamma induced increasing levels of tyrosine-phosphorylated STAT2 in M27(-) MCMV-infected cells that were essential for the antiviral potency of IFN-gamma. pM27 represents a new strategy for simultaneous evasions from types I and II IFNs, and it documents an unknown biological significance for STAT2 in antiviral IFN-gamma responses.
Asunto(s)
Proteínas Portadoras/inmunología , Proteínas de Unión al ADN/inmunología , Infecciones por Herpesviridae/inmunología , Interferones/inmunología , Muromegalovirus/inmunología , Transducción de Señal/inmunología , Transactivadores/inmunología , Proteínas Virales/inmunología , Animales , Proteínas Portadoras/genética , Células Cultivadas , Regulación de la Expresión Génica/inmunología , Infecciones por Herpesviridae/genética , Ratones , Ratones Noqueados , Muromegalovirus/genética , Receptores de Interferón/biosíntesis , Factor de Transcripción STAT2 , Transcripción Genética/inmunología , Activación Transcripcional/inmunología , Proteínas Virales/genéticaRESUMEN
The NK cell-activating receptor NKG2D interacts with three different cellular ligands, all of which are regulated by mouse cytomegalovirus (MCMV). We set out to define the viral gene product regulating murine UL16-binding protein-like transcript (MULT)-1, a newly described NKG2D ligand. We show that MCMV infection strongly induces MULT-1 gene expression, but surface expression of this glycoprotein is nevertheless completely abolished by the virus. Screening a panel of MCMV deletion mutants defined the gene m145 as the viral regulator of MULT-1. The MCMV m145-encoded glycoprotein turned out to be necessary and sufficient to regulate MULT-1 by preventing plasma membrane residence of MULT-1. The importance of MULT-1 in NK cell regulation in vivo was confirmed by the attenuating effect of the m145 deletion that was lifted after NK cell depletion. Our findings underline the significance of escaping MULT-1/NKG2D signaling for viral survival and maintenance.