IMPROVEMENT OF THE SPECIFIC INFECTIVITY OF THE RUBELLA-VIRUS (RUB) INFECTIOUS CLONE - DETERMINANTS OF CYTOPATHOGENICITY INDUCED BY RUB MAP TO THE NONSTRUCTURAL PROTEINS

A plasmid, Robo102, which contains a cDNA copy of the rubella virus (R UB) genomic RNA from which infectious transcripts can be synthesized i n vitro, was recently developed (C. Y. Wang, G, Dominguez, and T. K. F rey, J, Virol. 68:3550-3557, 1994), To increase the specific infectivi ty of Robo102 transcripts (similar to 5 plaques/10 mu g of transcripts ), a modified reverse transcription-PCR method was used to amplify nea rly 90% of the RUB genome in three fragments, which were then used to replace the corresponding fragments in Robo102. Replacement of a fragm ent covering nucleotides (nt) 5352 to 9759 of the RUB genome yielded a construct, Robo202, which produced highly infectious transcripts (10( 4) plaques/mu g, indicating the presence of an unrecognized deleteriou s mutation (or mutations) in this region of the Robo102 cDNA. Robo102 was based on the w-Therien strain of RUB, which forms opaque plaques i n Vero cells, while the PCR replacement fragments were generated from a variant, f-Therien, which produces clear plaques in Vero cells. Alth ough Robo202 contains over 4,000 nt from f-Therien, Robo202 virus prod uces opaque plaques, However, when the other two PCR fragments amplifi ed from f=Therien (nt 1 to 1723 and nt 2800 to 5352) were introduced i nto Robo202, the resulting construct, Robo302, yielded transcripts tha t produced a virus that formed clear plaques. This indicates that the determinants of plaque morphology map to the regions of the genome cov ered by these two fragments, both of which are in the nonstructural op en reading frame, Generation of Robo202/302 chimeras indicated that th e most 5' terminal fragment (nt 1 to 1723) had the greatest effect on plaque morphology. The plaque morphology was correlated with the abili ty; of the viruses to hill infected cells, The only difference at the molecular level detected among the viruses was that the more cytopathi c viruses produced more nonstructural proteins than did the less cytop athic viruses. This finding, as well as the mapping of the genetic det erminants to the region of the genome encoding these proteins, indicat es that the nonstructural proteins can mediate cell killing.