Effects of defined mutations in the 5' nontranslated region of rubella virus genomic RNA on virus viability and macromolecule synthesis.

The 5' end of the genomic RNA of rubella virus (RUB) contains a 14-nucleotide (nt) single-stranded leader (ss-leader) followed by a stem-and-loop structure [5'(+)SL] (nt 15 to 65), the complement of which at the 3' end of the minus-strand RNA [3'(-)SL] has been proposed to function as a promoter for synthesis of genomic plus strands. A second intriguing feature of the 5' end of the RUB genomic RNA is the presence of a short (17 codons) open reading frame (ORF) located between nt 3 and 54; the ORF encoding the viral nonstructural proteins (NSPs) initiates at nt 41 in an alternate translational frame. To address the functional significance of these features, we compared the 5'-terminal sequences of six different strains of RUB, with the result that the short ORF is preserved (although the coding sequence is not conserved) as is the stem part of both the 5'(+)SL and 3'(-)SL, while the upper loop part of both structures varies. Next, using Robo302, an infectious cDNA clone of RUB, we introduced 31 different mutations into the 5'-terminal noncoding region, and their effects on virus replication and macromolecular synthesis were examined. This mutagenesis revealed that the short ORF is not essential for virus replication. The AA dinucleotide at nt 2 and 3 is of critical importance since point mutations and deletions that altered or removed both of these nucleotides were lethal. None of the other mutations within either the ss-leader or the 5'(+)SL [and accordingly within the 3'(-)SL], including deletions of up to 15 nt from the 5'(+)SL and three different multiple-point mutations that lead to destabilization of the 5'(+)SL, were lethal. Some of the mutations within both ss-leader and the 5'(+)SL resulted in viruses that grew to lower titers than the wild-type virus and formed opaque and/or small plaques; in general mutations within the stem had a more profound effect on viral phenotype than did mutations in either the ss-leader or upper loop. Mutations in the 5'(+)SL, but not in the ss-leader, resulted in a significant reduction in NSP synthesis, indicating that this structure is important for efficient translation of the NSP ORF. In contrast, viral plus-strand RNA synthesis was unaffected by the 5'(+)SL mutations as well as the ss-leader mutations, which argues against the proposed function of the 3'(-)SL as a promoter for initiation of the genomic plus-strand RNA.