DEMONSTRATION OF THE SPECIFICITY OF POLIOVIRUS ENCAPSIDATION USING A NOVEL REPLICON WHICH ENCODES ENZYMATICALLY ACTIVE FIREFLY LUCIFERASE

The specificity of poliovirus encapsidation has been studied using a n ovel chimeric genome in which the gene encoding firefly luciferase has been substituted for the VP2-VP3-VP1 genes of the poliovirus capsid ( P1) gene. Transfection of RNA transcribed in vitro from this genome re sulted in a VP4-luciferase fusion protein which retained luciferase en zyme activity. Since the detection of enzyme activity was dependent up on replication of the transfected RNA genome, we refer to these genome s as replicons. The replicon encoding luciferase was encapsidated Upon transfection of the genomic RNA into cells previously infected with a recombinant vaccinia virus; W-P1, which encodes the poliovirus type 1 capsid proteins (P1). Infection of cells with each serial passage, fo llowed by analysis of luciferase enzyme activity, revealed that encaps idated replicons could be detected at the first passage with W-P1. Amp lification of the titer of encapsidated replicons occurred upon serial passage with W-P1, as evidenced by the high expression levels Of luci ferase enzyme activity following infection. serial passage of the luci ferase replicons with poliovirus type 1, 2, or 3 resulted in the Irans encapsidation into the type 1, 2, or 3 capsids, respectively. In cont rast, serial passage with bovine enterovirus, Coxsackievirus A21 or B3 , or enterovirus 70 did not result in Irans encapsidation, even though cc-infection of cells with the! replicon and different enteroviruses resulted ih high-level expression of luciferase. The results of this s tudy highlight the specificity of poliovirus encapsidation and point t o the use of encapsidated replicons encoding luciferase as a reagent f or dissecting elements of replication and encapsidation. (C) 1998 Acad emic Press.