Abstract
BackgroundSemliki 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.ResultsWe 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.ConclusionThe 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.
Highlights
Semliki Forest virus (SFV) has a positive strand RNA genome and infects different cells of vertebrates and invertebrates
Construction and viability of SFV4-based replicationcompetent vectors with duplicated SG promoters We have previously observed, using the SFV-based virus-like particles (VLPs) system, that expression of the protein of interest from a short 20/6 SG promoter of SFV4 was considerably weaker than that from different internal ribosomal entry site (IRES) elements [29]. This indicates that the minimal SG promoter for SFV4 is likely to be longer than the minimal 19/5 SG promoter for Sindbis virus (SINV)
Two sets of vectors with duplicated SG promoters, each consisting of four different constructs, were made and tested for their ability to express SFV4 ns proteins, structural proteins and inserted marker protein
Summary
Semliki Forest virus (SFV) has a positive strand RNA genome and infects different cells of vertebrates and invertebrates. SG-mRNA is generated by internal initiation at the SG-promoter that is located at the complementary minus-strand template. 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. The structural proteins, encoded by the 3' one-third of the genome, are translated from a subgenomic (SG) mRNA, which is generated by internal initiation from the SG promoter that is located on the complementary minus-strand template. The nonstructural (ns) polyprotein is translated directly from the viral genomic RNA. It is processed into individual components, the ns proteins nsP1–nsP4. Semliki Forest virus (SFV), Sindbis virus (SINV) and Venezuelan equine encephalitis virus (VEEV) are the best studied alphaviruses, and have been used for the development of gene expression systems [3,4,5]
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