RESUMEN
The rabies virus (RV) phosphoprotein P is a multifunctional protein involved in viral RNA synthesis and in counteracting host innate immune responses. We have previously shown that RV P gene expression levels can be regulated by using picornavirus internal ribosome entry site (IRES) elements. Here we exploited a particular feature of the foot-and-mouth disease virus (FMDV) IRES, namely, preferential initiation at a downstream initiation codon, to address the role of N-terminally truncated RV phosphoproteins usually generated in RV-infected cells through ribosomal leaky scanning. Recombinant RVs in which P synthesis was directed by the poliovirus or FMDV IRES produced full-length P (P1) or a truncated form (P2), as the dominant product, respectively. While the P2 overexpressing virus showed attenuated growth in interferon-incompetent cells, it was superior to the P1 overexpressing virus in preventing expression of host interferon-stimulated genes. This indicates that in RV infected cells the availability of the truncated P2 protein is critical for viral resistance to interferon.
Asunto(s)
Ingeniería Genética/métodos , Genoma Viral , Interferones/biosíntesis , Fosfoproteínas/genética , Biosíntesis de Proteínas , Isoformas de Proteínas/genética , Virus de la Rabia/genética , Rabia/virología , Proteínas Estructurales Virales/genética , Animales , Línea Celular , Codón Iniciador/genética , Cricetinae , ARN Polimerasas Dirigidas por ADN/genética , Virus de la Fiebre Aftosa/química , Virus de la Fiebre Aftosa/genética , Genes Reporteros , Interferones/inmunología , Luciferasas/análisis , Chaperonas Moleculares , Iniciación de la Cadena Peptídica Traduccional/genética , Fosfoproteínas/química , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Plásmidos , Poliovirus/química , Poliovirus/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Rabia/inmunología , Virus de la Rabia/inmunología , Virus de la Rabia/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Transfección , Proteínas Virales/genética , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/inmunología , Proteínas Estructurales Virales/metabolismoRESUMEN
Gene expression of nonsegmented negative-strand RNA viruses is regulated at the transcriptional level and relies on the canonical 5'-end-dependent translation of capped viral mRNAs. Here, we have used internal ribosome entry sites (IRES) from picornaviruses to control the expression level of the phosphoprotein P of the neurotropic rabies virus (RV; Rhabdoviridae), which is critically required for both viral replication and escape from the host interferon response. In a dual luciferase reporter RV, the IRES elements of poliovirus (PV) and human rhinovirus type 2 (HRV2) were active in a variety of cell lines from different host species. While a generally lower activity of the HRV2 IRES was apparent compared to the PV IRES, specific deficits of the HRV2 IRES in neuronal cell lines were not observed. Recombinant RVs expressing P exclusively from a bicistronic nucleoprotein (N)-IRES-P mRNA showed IRES-specific reduction of replication in cell culture and in neurons of organotypic brain slice cultures, an increased activation of the beta interferon (IFN-beta) promoter, and increased sensitivity to IFN. Intracerebral infection revealed a complete loss of virulence of both PV- and HRV2 IRES-controlled RV for wild-type mice and for transgenic mice lacking a functional IFN-alpha receptor (IFNAR(-/-)). The virulence of HRV2 IRES-controlled RV was most severely attenuated and could be demonstrated only in newborn IFNAR(-/-) mice. Translational control of individual genes is a promising strategy to attenuate replication and virulence of live nonsegmented negative-strand RNA viruses and vectors and to study the function of IRES elements in detail.