title: Development of a rubella virus vaccine expression vector: use of a picornavirus internal ribosome entry site increases stability of expression.
abstract: Rubella virus (RUB) is a small plus-strand RNA virus classified in the Rubivirus genus of the family Togaviridae. Live, attenuated RUB vaccines have been successfully used in vaccination programs for over 25 years, making RUB an attractive vaccine vector. In this study, such a vector was constructed using a recently developed RUB infectious cDNA clone (Robo). Using a standard strategy employed to produce expression and vaccine vectors with other togaviruses, the subgenomic promoter was duplicated to produce a recombinant construct (termed dsRobo) that expressed reporter genes such as chloramphenicol acetyltransferase and green fluorescent protein (GFP) under control of the second subgenomic promoter. However, expression of the reporter genes, as exemplified by GFP expression by dsRobo/GFP virus, was unstable during passaging, apparently due to homologous recombination between the subgenomic promoters leading to deletion of the GFP gene. To improve the stability of the vector, the internal ribosome entry site (IRES) of a picornavirus, encephalomyocarditis virus, was used instead of the second subgenomic promoter to eliminate homology. Construction was initiated by first replacing the subgenomic promoter in the parent Robo infectious clone with the IRES. Surprisingly, viable virus resulted; this virus did not synthesize a subgenomic RNA. The subgenomic promoter was then reintroduced in an orientation such that a single subgenomic RNA was produced, GFP was the initial gene on this RNA, while the RUB structural protein open reading frame was downstream and under control of the IRES element. GFP expression by this vector was significantly improved in comparison to dsRobo/GFP. This strategy should be applicable to increase the stability of other togavirus vectors.
authors.affiliation: Department of Biology, Georgia State University, Atlanta, Georgia 30303, USA.
authors.forename: K V
authors.initials: KV
authors.lastname: Pugachev
authors.forename: W P
authors.initials: WP
authors.lastname: Tzeng
authors.forename: T K
authors.initials: TK
authors.lastname: Frey
mesh_terms: D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D004686:Encephalomyocarditis virus / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012270:Ribosomes / Q000378:metabolism; D012411:Rubella Vaccine / Q000235:genetics*; D012412:Rubella virus / Q000235:genetics / Q000378:metabolism; D014709:Vero Cells
chemical_list: D018076:DNA, Complementary; D008164:Luminescent Proteins; D012411:Rubella Vaccine; D049452:Green Fluorescent Proteins; D015500:Chloramphenicol O-Acetyltransferase