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A plasmid, Robo102, which contains a cDNA copy of the rubella virus (RUB) genomic RNA from which infectious transcripts can be synthesized in vitro, was recently developed (C. Y. Wang, G. Dominguez, and T. K. Frey, J. Virol. 68:3550-3557, 1994). To increase the specific infectivity of Robo102 transcripts (approximately 5 plaques/10 microg of transcripts), a modified reverse transcription-PCR method was used to amplify nearly 90% of the RUB genome in three fragments, which were then used to replace the corresponding fragments in Robo102. Replacement of a fragment covering nucleotides (nt) 5352 to 9759 of the RUB genome yielded a construct, Robo202, which produced highly infectious transcripts (10(4) plaques/microg), indicating the presence of an unrecognized deleterious mutation (or mutations) in this region of the Robo102 cDNA. Robo102 was based on the w-Therien strain of RUB, which forms opaque plaques in Vero cells, while the PCR replacement fragments were generated from a variant, f-Therien, which produces clear plaques in Vero cells. Although Robo202 contains over 4,000 nt from f-Therien, Robo202 virus produces opaque plaques. However, when the other two PCR fragments amplified from f-Therien (nt 1 to 1723 and nt 2800 to 5352) were introduced into Robo202, the resulting construct, Robo302, yielded transcripts that produced a virus that formed clear plaques. This indicates that the determinants of plaque morphology map to the regions of the genome covered by these two fragments, both of which are in the nonstructural open reading frame. Generation of Robo202/302 chimeras indicated that the most 5' terminal fragment (nt. 1 to 1723) had the greatest effect on plaque morphology. The plaque morphology was correlated with the ability of the viruses to kill infected cells. The only difference at the molecular level detected among the viruses was that the more cytopathic viruses produced more nonstructural proteins than did the less cytopathic viruses. This finding, as well as the mapping of the genetic determinants to the region of the genome encoding these proteins, indicates that the nonstructural proteins can mediate cell killing.

journal of virology

Improvement of the specific infectivity of the rubella virus (RUB) infectious clone: determinants of cytopathogenicity induced by RUB map to the nonstructural proteins