In vitro replication of Sendai virus wild-type and defective interfering particle genome RNAs

Abstract

A system for studying the in vitro replication of the genome RNAs of Sendai virus and its defective interfering particle DI-H has been developed. Cytoplasmic extracts of baby hamster kidney cells infected with wild-type Sendai virus or coinfected with wild-type Sendai virus plus DI-H were prepared after lysolecithin treatment at 12 h postinfection. The extracts supported the transcription of six viral mRNAs as well as the replication of the Sendai virus 50S (wild-type) and 14S DI-H genome RNAs and their encapsidation into nucleocapsids in the absence of de novo protein synthesis. RNA replication in vitro represented more than 50% of total RNA synthesis, a relative level higher than that found in the infected cell. The proteins required for Sendai virus RNA replication were present in a soluble protein pool at the time of extract preparation. Depletion of the protein pool by prior treatment of infected cells with cycloheximide inhibited subsequent in vitro genome replication without affecting transcription. The cytoplasmic extract may be separated by high-speed centrifugation into two components: the Sendai virus wild-type and DI-H nucleocapsid templates containing the RNA and associated NP, L, and P proteins and the soluble protein fraction containing primarily the P, NP, and M viral proteins with trace amounts of the L, HN, Fo, and nonstructural C proteins. The isolated intracellular DI-H nucleocapsid template alone cannot replicate its RNA, but when recombined with the Sendai virus soluble protein fraction it catalyzes the replication and encapsidation of viral RNAs. The initiation of RNA replication in vitro can be demonstrated because detergent-disrupted purified DI-H virions replicate both positive- and negative-strand RNAs in the presence, but not in the absence, of the soluble protein fraction from an extract of infected cells.