Point mutations in the ICR2 motif of brome mosaic virus RNAs debilitate (+)-strand replication.

Sequences at the 5' termini of the genomic RNAs of brome mosaic virus (BMV) and other (+)-stranded RNA viruses have been shown (L.E. Marsh and T.C. Hall, 1987, Cold Spring Harbor Symp. Quant. Biol. 52, 331-341) to resemble the ICRs 1 and 2 (A and B boxes) of tRNA genes, with the complementary sequences at the 3' termini of the (-) strands resembling the ICR2 motif of methionine initiator tRNA genes (L.E. Marsh, G.P. Pogue, and T.C. Hall, 1989, Virology 172, 415-427). In order to examine the role of these sequences in viral replication, point mutations have been introduced into the ICR2-like sequence of a BMV RNA-2 deletion mutant, pRNA delta M/S (parasitic RNA), that does not encode a functional viral protein but replicates in the presence of genomic RNA-1 and -2. Single-base substitutions introduced at positions A7 or T8 of the (+)-sense ICR2-like motif reduced pRNA delta M/S replication by 70-82%, the primary effect being shown by kinetic analyses to be debilitation of (+)-strand synthesis. Whether these motifs act in their (+)-sense orientation in a manner analogous to tRNA genes or through the tRNA(Meti)-like sequence on the 3' (-) strand remains to be determined, but the data clearly demonstrate that the base composition within the ICR-like region of BMV RNAs contributes greatly to (+)-strand promoter function.