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

The S3L2-ACUucc pk mutant, in which frameshifting was reduced nearly 3-fold, was viable. However, while the mutant replicated to levels similar to the wild-type virus at later times pi, the inability to dimerize in infected Vero cells significantly changed the viral growth kinetics, RNA species and replicase protein levels. The fact that a silent codon change in the Loop 2 of SARS-CoV reproducibly affected the levels of gRNA and sgRNA strongly suggests that dimer formation occurs in the cellular environment and that loop-loop kissing interactions involving Stem 3 of the pseudoknot are important for accumulation of sgRNA. The in vitro findings of reduced total RNA levels and ORF1a/b protein following infection leading to a lag in replication are consistent with reduced levels of ORF1b translation products as a result of the 3-fold reduction in -1 PRF. The downregulation of the ORF1a gene product nsp1 is likely a result of the decrease in the amount of gRNA in the infected cells.