RESUMEN
Deletion or truncation of NS1, the principal IFN antagonist of influenza viruses, leads to increased IFN induction during influenza virus infection. We have studied activation of the IFN induction cascade by both wild-type and NS1-defective viruses at the single-cell level using a cell line expressing GFP under the control of the IFN-ß promoter and by examining MxA expression. The IFN-ß promoter was not activated in all infected cells even during NS1-defective virus infections. Loss of NS1 expression is therefore insufficient per se to induce IFN in an infected cell, and factors besides NS1 expression status must dictate whether the IFN response is activated. The IFN response was efficiently stimulated in these cells following infection with other viruses; the differential IFN response we observe with influenza viruses is therefore not cell specific but is likely due to differences in the nature of the infecting virus particles and their subsequent replication.
Asunto(s)
Virus de la Influenza A/fisiología , Gripe Humana/inmunología , Gripe Humana/virología , Interferón beta/genética , Regiones Promotoras Genéticas , Activación Transcripcional , Proteínas no Estructurales Virales/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Virus de la Influenza A/genética , Gripe Humana/genética , Proteínas de Resistencia a Mixovirus/genética , Análisis de la Célula Individual , Proteínas no Estructurales Virales/genética , Internalización del Virus , Replicación ViralRESUMEN
Preparations of parainfluenza virus 5 (PIV5) that are potent activators of the interferon (IFN) induction cascade were generated by high-multiplicity passage in order to accumulate defective interfering virus genomes (DIs). Nucleocapsid RNA from these virus preparations was extracted and subjected to deep sequencing. Sequencing data were analyzed using methods designed to detect internal deletion and "copyback" DIs in order to identify and characterize the different DIs present and to approximately quantify the ratio of defective to nondefective genomes. Trailer copybacks dominated the DI populations in IFN-inducing preparations of both the PIV5 wild type (wt) and PIV5-VΔC (a recombinant virus that does not encode a functional V protein). Although the PIV5 V protein is an efficient inhibitor of the IFN induction cascade, we show that nondefective PIV5 wt is unable to prevent activation of the IFN response by coinfecting copyback DIs due to the interfering effects of copyback DIs on nondefective virus protein expression. As a result, copyback DIs are able to very rapidly activate the IFN induction cascade prior to the expression of detectable levels of V protein by coinfecting nondefective virus.
Asunto(s)
Virus Defectuosos/genética , Genoma Viral , Infecciones por Rubulavirus/inmunología , Infecciones por Rubulavirus/virología , Rubulavirus/genética , Animales , Línea Celular , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Interferones/genética , Interferones/inmunología , Infecciones por Rubulavirus/genética , Proteínas Virales/genéticaRESUMEN
Conflicting reports exist regarding the requirement for virus replication in interferon (IFN) induction by paramyxoviruses. Our previous work has demonstrated that pathogen-associated molecular patterns capable of activating the IFN-induction cascade are not normally generated during virus replication, but are associated instead with the presence of defective interfering (DI) viruses. We demonstrate here that DIs of paramyxoviruses, including parainfluenza virus 5, mumps virus and Sendai virus, can activate the IFN-induction cascade and the IFN-ß promoter in the absence of virus protein synthesis. As virus protein synthesis is an absolute requirement for paramyxovirus genome replication, our results indicate that these DI viruses do not require replication to activate the IFN-induction cascade.
Asunto(s)
Interferón beta/biosíntesis , Interferón beta/genética , Paramyxoviridae/inmunología , Paramyxoviridae/fisiología , Regiones Promotoras Genéticas , Activación Transcripcional , Replicación Viral , Animales , Línea Celular , Virus Defectuosos/genética , Virus Defectuosos/inmunología , Humanos , Paramyxoviridae/genética , Rubulavirus , Proteínas Virales/biosíntesisRESUMEN
It is generally thought that pathogen-associated molecular patterns (PAMPs) responsible for triggering interferon (IFN) induction are produced during virus replication and, to limit the activation of the IFN response by these PAMPs, viruses encode antagonists of IFN induction. Here we have studied the induction of IFN by parainfluenza virus type 5 (PIV5) at the single-cell level, using a cell line expressing GFP under the control of the IFN-ß promoter. We demonstrate that a recombinant PIV5 (termed PIV5-VΔC) that lacks a functional V protein (the viral IFN antagonist) does not activate the IFN-ß promoter in the majority of infected cells. We conclude that viral PAMPs capable of activating the IFN induction cascade are not produced or exposed during the normal replication cycle of PIV5, and suggest instead that defective viruses are primarily responsible for inducing IFN during PIV5 infection in this system.
Asunto(s)
Interferón beta/antagonistas & inhibidores , Interferón beta/genética , Regiones Promotoras Genéticas , Rubulavirus/fisiología , Proteínas Virales/fisiología , Animales , Línea Celular , Chlorocebus aethiops , Virus Defectuosos/genética , Virus Defectuosos/fisiología , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Immunoblotting , Interferón beta/metabolismo , Mutación , Rubulavirus/genética , Células Vero , Proteínas Virales/genética , Replicación ViralRESUMEN
The infection of cells by RNA viruses is associated with the recognition of virus PAMPs (pathogen-associated molecular patterns) and the production of type I interferon (IFN). To counter this, most, if not all, RNA viruses encode antagonists of the IFN system. Here we present data on the dynamics of IFN production and response during developing infections by paramyxoviruses, influenza A virus and bunyamwera virus. We show that only a limited number of infected cells are responsible for the production of IFN, and that this heterocellular production is a feature of the infecting virus as opposed to an intrinsic property of the cells.
