A conserved hairpin structure in Alfamovirus and Bromovirus subgenomic promoters is required for efficient RNA synthesis in vitro

The coat protein gene in RNA 3 of alfalfa mosaic virus (AMV; genus Alfamovi rus, family Bromoviridae) is translated from the subgenomic RNA 4. Analysis of the subgenomic promoter (sgp) in minus-strand RNA 3 showed that a seque nce of 37 nt upstream of the RNA 4 start site (nt +1) was sufficient for fu ll sgp activity in an in vitro assay with the purified viral RNA-dependent RNA-polymerase (RdRp). The sequence of nt -6 to -29 could be folded into a potential hairpin structure with a loop represented by nt -16, -17, and -18 , and a bulge involving nt -23. By introducing mutations that disrupted bas e pairing and compensatory mutations that restored base pairing, it was sho wn that base pairing in the top half of the putative stem (between the loop and bulge) was essential for sgp activity, whereas base pairing in the bot tom half of the stem was less stringently required. Deletion of the bulged residue A-23 or mutation of this residue into a C strongly reduced sgp acti vity, but mutation of A-23 into U or G had little effect on sgp activity. M utation of loop residues A-16 and A-17 affected sgp activity, whereas mutat ion of U-18 did not. Using RNA templates corresponding to the sgp of brome mosaic virus (BMV; genus Bromovirus, family Bromoviridae) and purified BRV RdRp, evidence was obtained indicating that also in BMV RNA a triloop hairp in structure is required for sgp activity.