Requirement for ICR-like sequences in the replication of brome mosaic virus genomic RNA.

Previous studies using a brome mosaic virus (BMV) RNA-2 deletion mutant (pRNA-2 M/S) and additional derivatives as reporters established that viral sequences resembling internal control regions (ICRs) 1 and 2 of tRNA gene promoters are vital to (+)-strand replication in protoplasts. Transfer of these mutations to genomic RNA-2 and functional analysis in protoplast, local lesion, and systemic infections revealed a sequence-specific requirement for bases within the ICR2-like motif. Despite the low (generally less than 20% of wild-type) and sometimes undetectable levels of replication of these RNA-2 mutants, sufficient p2a protein was produced to support at least modest levels of RNA-1, -3 and -4 replication in protoplasts. However, only those RNA-2 ICR2 mutants supporting substantial replication of the viral genome in protoplasts were capable of establishing local lesions in Chenopodium hybridum and systemic infections in barley, further establishing the essential role of the ICR-like sequences in viral infectivity. Upon passage through a second set of barley plants, accumulation patterns for progeny from inocula containing certain RNA-2 mutants paralleled those from wild-type inocula, indicating repair of the introduced mutations. RNA stability and translatability were shown to be unaffected by the introduced mutations. BMV RNA-3 contains several ICR-like sequences, each of which was individually deleted. Whereas deletion of the 5'-terminal ICR2-like motif had little effect on RNA-3 accumulation in protoplasts or local lesion formation, it debilitated systemic spread in barley. Deletion of an internal ICR2-like motif at position 1100 decreased (+):(-) strand asymmetry from greater than 100:1 to 14:1, reduced RNA-3 replication in protoplasts to less than 15% of wild-type, and abolished local lesion and systemic infectivity.