RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus

Ishimaru, Daniella; Plant, Ewan P.; Sims, Amy C.; Yount, Boyd L.; Roth, Braden M.; Eldho, Nadukkudy V.; Pérez-Alvarado, Gabriela C.; Armbruster, David W.; Baric, Ralph S.; Dinman, Jonathan D.; Taylor, Deborah R.; Hennig, Mirko

To determine the importance of L2-mediated selfassociation for SARS-CoV -1 PRF, several pseudoknot variants were subjected to a dual luciferase reporter-based frameshifting analysis as previously described ( Figure 8A ) (1, 39) . A Loop 2 ACUucc silent (serine codon) mutation was made to investigate the role of the palindrome (S3L2-ACUucc; Figure 1D ) in promoting -1 PRF. Phylogenetic analysis indicated that the sequence UCC is present at a (1). Dual luciferase measurements of -1 PRF from the S3L2-ACUucc pk construct showed a 63% reduction in frameshift stimulation compared with wild-type pk (5.7% versus 15.0%, Figure 8A ). Next, we tested two S3 mutants for frameshift stimulation in the luciferase assay. S3-cuug ( Figure 1E ), which we showed to readily self-associate ( Figure 3A, lane 2) , demonstrated a nominal increase in frameshift frequency (17.5%), while a S3 truncation of the same construct (S3-2bp-cuug, Figure 1F ) exhibited near wild-type efficiency (15.2%). It is important to note that the previously characterized S3-deletion ÁS3 pk construct maintained dimerization capability albeit with reduced efficiency (Figures 3B and C, lane 3) . Our results are consistent with previous analyses by three different groups that have shown mutations to the third stem of a three-stemmed pseudoknot to have less impact on frameshifting efficiency than mutations to Stem 2 (1,2,6,7). However, our data indicate that silent codon changes to Loop 2 that disrupt the palindrome play a role in regulating the frequency of frameshifting.