COMPLEMENTATION OF A POLIOVIRUS DEFECTIVE GENOME BY A RECOMBINANT VACCINIA VIRUS WHICH PROVIDES POLIOVIRUS P1 CAPSID PRECURSOR IN TRANS

Defective interfering (DI) RNA genomes of poliovirus which contain in- frame deletions in the P1 capsid protein-encoding region have been des cribed. DI genomes are capable of replication and can be encapsidated by capsid proteins provided in trans from wild-type poliovirus. In thi s report, we demonstrate that a previously described poliovirus DI gen ome (K. Hagino-Yamagishi and A. Nomoto, J. Virol. 63:5386-5392, 1989) can be complemented by a recombinant vaccinia virus, VVP1 (D. C. Ansar di, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991), whi ch expresses the poliovirus capsid precursor polyprotein, P1. Stocks o f defective polioviruses were generated by transfecting in vitro-trans cribed defective genome RNA derived from plasmid pSM1 (T7)1 into HeLa cells infected with VVP1 and were maintained by serial passage in the presence of VVP1. Encapsidation of the defective poliovirus genome was demonstrated by characterizing poliovirus-specific protein expression in cells infected with preparations of defective poliovirus and by No rthern (RNA) blot analysis of poliovirus-specific RNA incorporated int o defective poliovirus particles. Cells infected with preparations of defective poliovirus expressed poliovirus protein 3CD but did not expr ess capsid proteins derived from a full-length P1 precursor. Polioviru s-specific RNA encapsidated in viral particles generated in cells coin fected with VVP1 and defective poliovirus migrated slightly faster on formaldehyde-agarose gels than wild-type poliovirus RNA, demonstrating maintenance of the genomic deletion. By metabolic radiolabeling with [S-35]methionine-cysteine, the defective poliovirus particles were sho wn to contain appropriate mature-virion proteins. This is the first re port of the generation of a pure population of defective polioviruses free of contaminating wild-type poliovirus. We demonstrate the use of this recombinant vaccinia virus-defective poliovirus genome complement ation system for studying the effects of a defined mutation in the P1 capsid precursor on virus assembly. Following removal of residual VVP1 from defective poliovirus preparations, processing and assembly of po liovirus capsid proteins derived from a nonmyristylated P1 precursor e xpressed by a recombinant vaccinia virus, VVP1 myr- (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 66:4556-4563, 1992), in cells coinfected with defective poliovirus were analyzed. Capsid proteins ge nerated from nonmyristylated P1 did not assemble detectable levels of mature virions but did assemble, at low levels, into empty capsids. Th ese results contrast with an earlier study from our laboratory, in whi ch assembly of processed capsid proteins generated from nonmyristylate d P1 was not observed with cells coinfected with a recombinant vaccini a virus expressing poliovirus 3CD protease, and suggest that component s of a poliovirus infection, such as poliovirus-induced membrane vesic les and poliovirus genomic RNA, facilitate the early stages of poliovi rus capsid assembly.