CHARACTERIZATION OF DEFECTIVE-INTERFERING RNAS OF RUBELLA-VIRUS GENERATED DURING SERIAL UNDILUTED PASSAGE

During serial undiluted passage of rubella virus (RUB) in Vero cells, two species of defective-interfering (DI) RNAs of approximately 7000 a nd 800 nucleotides (nts) in length were generated (Frey, T. K., and He mphill, M. L., Virology 164, 22-29, 1988). In this study, these DI RNA s were characterized by molecular cloning, hybridization with probes o f defined sequence, and primer extension. The 7000-nt DI RNA species w ere found to be authentic DI RNAs which contain a single 2500- to 2700 -nt deletion in the structural protein open reading frame (ORF) region of the genome. The 800-nt RNAs were found to be subgenomic DI RNAs sy nthesized from the large DI RNA templates. Analysis of the extent of t he deletions using a reverse-transcription-PCR protocol revealed that the 3' end of the deletions did not extend beyond the 3' terminal 244 nts of the genome. The 5' end of the deletions did not extend into the nonstructural protein ORF; however, DI RNAs in which the subgenomic s tart site was deleted were present. Following serial undiluted passage of seven independent stocks of RUB, this was the only pattern of DI R NAs generated. DI RNAs of 2000 to 3000 nt in length were the majority DI RNA species in a persistently infected line of Vero cells, showing that other types of RUB DI RNAs can be generated and selected. However , when supernatant from the persistently infected cells was passaged, the only DI RNAs present after two passages were 7000 nts in length, i ndicating that this species has a selective advantage over other types of DI RNAs during serial passage. (C) 1995 Academic Press, Inc.