Rubella virus DI RNAs and replicons: Requirement for nonstructural proteins acting in cis for amplification by helper virus

A rubella virus (RUB) replicon was constructed by replacing the 3' proximal structural protein ORF (SP-ORF) in Robo402, a RUB infectious cDNA clone, w ith a reporter gene, green fluorescent protein (GFP). This replicon, RUBrep /GFP, mimics naturally occurring RUB defective-interfering (DI) RNAs genera ted during serial undiluted passage that maintain the 5' proximal nonstruct ural protein ORF (NS-ORF) but contain deletions in the SP-ORF Following tra nsfection of Vero cells with in vitro RNA transcripts from RUBrep/GFP, repl icon replication occurred and the replicon was amplified and spread to othe r cells in the presence of standard helper virus. GFP expression was a much more sensitive indicator of replicon replication than was Northern analysi s to detect replicon-specific RNAs. Most of a series of RUBrep/GFP construc ts with deletions in the NS-ORF not only were incapable of self-replication , but were not amplified by standard helper virus. The only exception was a construct with an in-frame deletion between two Not1 sites that removed nu cleotides 1685-2192 of the genome; this construct did not express GFP by it self, but did express GFP in the presence of standard helper RUB and was sp read to other cells. Thus, with the exception of this region, the NS-ORF is required in cis for amplification of RUB replicons by standard helper viru s, explaining the selection of DI RNAs that maintain the NS-ORF. Surprising ly, when the Not1 deletion was introduced into Robo402, a viable virus resu lted that replicated only threefold less efficiently than did Robo402 virus . Thus, the Noti region of the NS-ORF is not necessary for virus replicatio n. This deletion covers a region of the NS-ORF without predicted function, which therefore may function as a spacer or hinge between functional domain s. Nevertheless, it was an unexpected finding that a small virus such as RU B could dispense with similar to 10% of its genome. (C) 2001 Academic Press .