RESUMEN
BACKGROUND: Semliki Forest virus (SFV) has a positive strand RNA genome and infects different cells of vertebrates and invertebrates. The 5' two-thirds of the genome encodes non-structural proteins that are required for virus replication and synthesis of subgenomic (SG) mRNA for structural proteins. SG-mRNA is generated by internal initiation at the SG-promoter that is located at the complementary minus-strand template. Different types of expression systems including replication-competent vectors, which represent alphavirus genomes with inserted expression units, have been developed. The replication-competent vectors represent useful tools for studying alphaviruses and have potential therapeutic applications. In both cases, the properties of the vector, such as its genetic stability and expression level of the protein of interest, are important. RESULTS: We analysed 14 candidates of replication-competent vectors based on the genome of an SFV4 isolate that contained a duplicated SG promoter or an internal ribosomal entry site (IRES)-element controlled marker gene. It was found that the IRES elements and the minimal -21 to +5 SG promoter were non-functional in the context of these vectors. The efficient SG promoters contained at least 26 residues upstream of the start site of SG mRNA. The insertion site of the SG promoter and its length affected the genetic stability of the vectors, which was always higher when the SG promoter was inserted downstream of the coding region for structural proteins. The stability also depended on the conditions used for vector propagation. A procedure based on the in vitro transcription of ligation products was used for generation of replication-competent vector-based expression libraries that contained hundreds of thousands of different genomes, and maintained genetic diversity and the ability to express inserted genes over five passages in cell culture. CONCLUSION: The properties of replication-competent vectors of alphaviruses depend on the details of their construction. In the case of SFV4, such vectors should contain the SG promoter with structural characteristics for this isolate. The main factor for instability of SFV4-based replication-competent vectors was the deletion of genes of interest, since the resulting shorter genomes had a growth advantage over the original vector.
Asunto(s)
Regulación Viral de la Expresión Génica , Vectores Genéticos , Virus de los Bosques Semliki/genética , Virus de los Bosques Semliki/fisiología , Replicación Viral/fisiología , Regiones no Traducidas 3'/genética , Animales , Línea Celular , Cricetinae , Eliminación de Gen , Vectores Genéticos/genética , Vectores Genéticos/fisiología , Genoma Viral , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/biosíntesis , ARN Viral/genética , ARN Viral/metabolismo , Ribosomas/metabolismo , Virus de los Bosques Semliki/metabolismo , Transcripción Genética , Transfección , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismoRESUMEN
In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.
Asunto(s)
Culicidae/virología , Interferencia de ARN , Virus de los Bosques Semliki/genética , Animales , Línea Celular , Cricetinae , Regulación Viral de la Expresión Génica , ARN Viral/genética , Replicación ViralRESUMEN
Semliki Forest virus (SFV, genus Alphavirus) has a broad host range, high efficiency of viral protein expression, and the ability to stimulate an immune response. These properties have made SFV an attractive tool for development of expression vectors, and plasmid clones containing cDNA of the SFV genome often are used. However, instability of these plasmids resulting from cryptic expression of SFV envelope proteins in Escherichia coli represents a problem both for the development of SFV-based vectors and for SFV research. In this study, an infectious plasmid of SFV, pCMV-SFV4, was constructed; its toxic effect was eliminated by intron insertion in the capsid protein encoding region. When transfected into mammalian cells, the plasmid clone was highly infectious and produced virus with properties identical to those of wild-type SFV. The inserted intron was efficiently and properly removed from the RNA genome of SFV. Therefore, this novel and stabilized infectious SFV plasmid represents a superior tool for basic studies of SFV as well as for biotechnological applications.
