Stimulation of stop codon readthrough: frequent presence of an extended 3' RNA structural element

Firth, Andrew E.; Wills, Norma M.; Gesteland, Raymond F.; Atkins, John F.

We have shown that the stimulatory elements for efficient RT in VEEV and probably also SINV include not just the immediately 3 0 -adjacent nucleotides, but also a stem-loop structure that spans $140 nt 3 0 of the stop codon. Computational analyses provide strong evidence that similar structures are relevant for RT in several other alphaviruses, and in plant viruses where RT occurs at a UGA codon. Although this RNA structure is clearly not essential for some level of RT to occur in some systems [as in many previous analyses the predicted WT structure was not present (32, 33, 74) and, in our own experiments, $1% RT was achieved in tissue culture without the WT structure], it does have a pronounced stimulatory effect on RT efficiency (3-to 4-fold for SINV, 10-to 14-fold for VEEV). As with the gammaretrovirus 3 0 pseudoknot, the precise mechanism by which the stem-loop affects RT remains to be determined. Possibilities include direct interaction with the ribosome (including pausing and/or promotion of conformational changes in the ribosome); provision of a physical block that preferentially occludes release factor from the A-site in favour of tRNAs; or an indirect action via some trans-acting factor. The function of the RNA structure may simply be to achieve a higher RT level that is optimal for the virus. Alternatively, the structure may provide a regulatory mechanism, perhaps allowing different RT levels to be achieved in different hosts or at different stages in the viral cycle.