RESUMEN
UNLABELLED: Interferon beta (IFN-ß) is a key component of cellular innate immunity in mammals, and it constitutes the first line of defense during viral infection. Studies with cultured cells previously showed that almost all nucleated cells are able to produce IFN-ß to various extents, but information about the in vivo sources of IFN-ß remains incomplete. By applying immunohistochemistry and employing conditional-reporter mice that express firefly luciferase under the control of the IFN-ß promoter in either all or only distinct cell types, we found that astrocytes are the main producers of IFN-ß after infection of the brain with diverse neurotropic viruses, including rabies virus, Theiler's murine encephalomyelitis virus, and vesicular stomatitis virus. Analysis of a panel of knockout mouse strains revealed that sensing of viral components via both RIG-I-like helicases and Toll-like receptors contributes to IFN induction in the infected brain. A genetic approach to permanently mark rabies virus-infected cells in the brain showed that a substantial number of astrocytes became labeled and, therefore, must have been infected by the virus at least transiently. Thus, our results strongly indicate that abortive viral infection of astrocytes can trigger pattern recognition receptor signaling events which result in secretion of IFN-ß that confers antiviral protection. IMPORTANCE: Previous work indicated that astrocytes are the main producers of IFN after viral infection of the central nervous system (CNS), but it remained unclear how astrocytes might sense those viruses which preferentially replicate in neurons. We have now shown that virus sensing by both RIG-I-like helicases and Toll-like receptors is involved. Our results further demonstrate that astrocytes get infected in a nonproductive manner under these conditions, indicating that abortive infection of astrocytes plays a previously unappreciated role in the innate antiviral defenses of the CNS.
Asunto(s)
Astrocitos/inmunología , Encéfalo/inmunología , Encéfalo/virología , Interferón beta/metabolismo , Virus de la Rabia/inmunología , Theilovirus/inmunología , Vesiculovirus/inmunología , Animales , Fusión Artificial Génica , Astrocitos/virología , Perfilación de la Expresión Génica , Genes Reporteros , Inmunohistoquímica , Luciferasas/análisis , Luciferasas/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Inmunológicos/metabolismo , Transducción de SeñalRESUMEN
During viral infections cellular gene expression is subject to rapid alterations induced by both viral and antiviral mechanisms. In this study, we applied metabolic labeling of newly transcribed RNA with 4-thiouridine (4sU-tagging) to dissect the real-time kinetics of cellular and viral transcriptional activity during lytic murine cytomegalovirus (MCMV) infection. Microarray profiling on newly transcribed RNA obtained at different times during the first six hours of MCMV infection revealed discrete functional clusters of cellular genes regulated with distinct kinetics at surprising temporal resolution. Immediately upon virus entry, a cluster of NF-κB- and interferon-regulated genes was induced. Rapid viral counter-regulation of this coincided with a very transient DNA-damage response, followed by a delayed ER-stress response. Rapid counter-regulation of all three clusters indicated the involvement of novel viral regulators targeting these pathways. In addition, down-regulation of two clusters involved in cell-differentiation (rapid repression) and cell-cycle (delayed repression) was observed. Promoter analysis revealed all five clusters to be associated with distinct transcription factors, of which NF-κB and c-Myc were validated to precisely match the respective transcriptional changes observed in newly transcribed RNA. 4sU-tagging also allowed us to study the real-time kinetics of viral gene expression in the absence of any interfering virion-associated-RNA. Both qRT-PCR and next-generation sequencing demonstrated a sharp peak of viral gene expression during the first two hours of infection including transcription of immediate-early, early and even well characterized late genes. Interestingly, this was subject to rapid gene silencing by 5-6 hours post infection. Despite the rapid increase in viral DNA load during viral DNA replication, transcriptional activity of some viral genes remained remarkably constant until late-stage infection, or was subject to further continuous decline. In summary, this study pioneers real-time transcriptional analysis during a lytic herpesvirus infection and highlights numerous novel regulatory aspects of virus-host-cell interaction.
Asunto(s)
Regulación Viral de la Expresión Génica , Infecciones por Herpesviridae/genética , Interacciones Huésped-Patógeno/genética , Muromegalovirus/genética , Animales , Perfilación de la Expresión Génica/métodos , Genes Virales/genética , Infecciones por Herpesviridae/virología , Ratones , Análisis por Micromatrices , Familia de Multigenes/genética , Muromegalovirus/patogenicidad , Células 3T3 NIH , Regiones Promotoras Genéticas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genética , Transcripción Genética/genéticaRESUMEN
The rabies virus (RV) phosphoprotein (P) is a type I interferon (IFN) antagonist preventing both transcriptional induction of IFN and IFN-mediated JAK/STAT signaling. In addition, P is an essential cofactor of the viral polymerase and is required for encapsidation of viral RNA into nucleoprotein during replication. By site-directed mutagenesis, we have identified a domain of P required for efficient inhibition of IFN induction. Phosphoproteins lacking amino acids (aa) 176 to 181, 182 to 186, or 176 to 186 were severely compromised in counteracting phosphorylation of IRF3 and IRF7 by TBK1 or IKKi while retaining the full capacity of preventing nuclear import of activated STATs and of supporting virus transcription and replication. Recombinant RV carrying the mutated phosphoproteins (the SAD ΔInd1, SAD ΔInd2, and SAD ΔInd1/2 viruses) activated IRF3 and beta IFN (IFN-ß) transcription in infected cells but still blocked STAT-mediated expression of IFN-stimulated genes. Due to a somewhat higher transcription rate, the SAD ΔInd1 virus activated IRF3 more efficiently than the SAD ΔInd2 virus. After intracerebral injection into mouse brains at high doses, the SAD ΔInd1 virus was completely apathogenic for wild-type (wt) mice, while the SAD ΔInd2 virus was partially attenuated and caused a slower progression of lethal rabies than wt RV. Neurovirulence of IFN-resistant RV thus correlates with the capacity of the virus to prevent activation of IRF3 and IRF7.
Asunto(s)
Factor 3 Regulador del Interferón/antagonistas & inhibidores , Factor 7 Regulador del Interferón/antagonistas & inhibidores , Interferón beta/antagonistas & inhibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Virus de la Rabia/inmunología , Virus de la Rabia/patogenicidad , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismo , Animales , Encéfalo/patología , Encéfalo/virología , Línea Celular , Modelos Animales de Enfermedad , Femenino , Quinasa I-kappa B , Masculino , Ratones , Ratones Transgénicos , Chaperonas Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas , Rabia/patología , Rabia/virología , Virus de la Rabia/genética , Análisis de Supervivencia , VirulenciaRESUMEN
Rabies is a zoonosis still claiming more than 50 000 human deaths per year. Typically, human cases are due to infection with rabies virus, the prototype of the Lyssavirus genus, but sporadic cases of rabies-like encephalitis caused by other lyssaviruses have been reported. In contrast to rabies virus, which has an extremely broad host range including many terrestrial warm-blooded animals, rabies-related viruses are associated predominantly with bats and rarely infect terrestrial species. In spite of a very close genetic relationship of rabies and rabies-related viruses, the factors determining the limited host range of rabies-related viruses are not clear. In the past years the importance of viral countermeasures against the host type I interferon system for establishment of an infection became evident. The rabies virus phosphoprotein (P) has emerged as a critical factor required for paralysing the signalling cascades leading to transcriptional activation of interferon genes as well as interferon signalling pathways, thereby limiting expression of antiviral and immune stimulatory genes. Comparative studies would be of interest in order to determine whether differential abilities of the lyssavirus P proteins contribute to the restricted host range of lyssaviruses.
Asunto(s)
Interferones/inmunología , Lyssavirus/fisiología , Infecciones por Rhabdoviridae/inmunología , Animales , Interacciones Huésped-Patógeno , Humanos , Lyssavirus/clasificación , Lyssavirus/inmunología , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Rabia/inmunología , Rabia/virología , Virus de la Rabia/clasificación , Virus de la Rabia/inmunología , Virus de la Rabia/fisiología , Infecciones por Rhabdoviridae/virología , Factores de Transcripción STAT/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Rabies is among the longest known and most dangerous and feared infectious diseases for humans and animals and still is responsible for tenth of thousands of human deaths per year. The rabies virus (RABV) is a rather atypical member of the Rhabdoviridae family as it has completely adapted during evolution to warm-blooded hosts and is directly transmitted between them, whereas most other rhabdoviruses are transmitted by insect vectors. The virus is also unique with respect to its extremely broad host species range and a very narrow host organ range, namely its strict neurotropism. It is becoming increasingly clear that the host innate immune system, particularly the type I interferon system, and the viral counteractions profoundly shape this virus-host relationship. In the past few years, exciting new insight was obtained on how viruses are sensed by innate immune receptors, how the downstream signaling networks for activation of interferon are working, and how viruses can interfere with the system. While RABV 5'-triphosphate RNAs were identified as the major pathogen-associated molecular pattern sensed by cytoplasmic RIG-I-like receptors (RLR), the RABV phosphoprotein (P) has emerged as a potent multifunctional antagonist able to counteract the signaling cascades leading to transcriptional activation of interferon genes as well as interferon signaling pathways, thereby limiting expression of antiviral and immune-stimulatory genes.
Asunto(s)
Interacciones Huésped-Patógeno , Evasión Inmune , Interferones/antagonistas & inhibidores , Interferones/inmunología , Virus de la Rabia/inmunología , Virus de la Rabia/patogenicidad , Animales , Humanos , Chaperonas Moleculares , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Proteínas Estructurales Virales/inmunología , Proteínas Estructurales Virales/metabolismo , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismoRESUMEN
The family Rhabdoviridae contains important pathogens of humans, livestock, and crops, including the insect-transmitted vesicular stomatitis virus (VSV) and the neurotropic rabies virus (RV), which is directly transmitted between mammals. In spite of a highly similar organization of RNA genomes, proteins, and virus particles, cell biology of VSV and RV is divergent in several aspects, particularly with respect to their interplay with the cellular host defense. While infection with both rhabdoviruses is recognized via viral triphosphate RNAs by the cytoplasmic RNA helicase/translocase RIG-I, the viral counteractions to limit the response are contrasting. VSV infection is characterized by a rapid general shutdown of host gene expression and severe cytopathic effects, due to multiple activities of the matrix (M) protein affecting host polymerase functions and mRNA nuclear export, and by rapid and high-level virus replication. In contrast, RV spread and transmission relies on preserving the integrity of host cells, particularly of neurons. While a general cell shutdown by RV M is not observed, RV phosphoprotein (P) has developed independent functions to interfere with activation of IRFs and with STAT signaling. The molecular mechanisms employed are different from those of the paramyxovirus P gene products serving similar functions, and illustrate evolution of IFN antagonists to specifically support virus survival in the natural niches.