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

Having shown that the SARS-CoV pseudoknot dimerizes in vitro and alters frameshifting in Vero cells, we asked whether the ability to self-associate is important for viral propagation. Site-directed mutagenesis was used to introduce the Loop 2 ACUucc silent mutation into SARS subclone D (40) , and the recombinant virus mutants were recovered from full-length virus transcripts. The observed effects should therefore reflect changes related to RNA structure within the frameshifting signal, rather than modifying the encoded proteins. To determine the effects on viral growth kinetics, stocks of rescued wild-type SARS-CoV and S3L2-ACUucc pk (multiplicity of infection (MOI) of 1) were used to infect VeroE6 cells, media was harvested at 0, 5 and 8 h pi and tested by plaque assay. Viral titers for SARS-CoV increased by wild-type pk ΔS3 pk S3L2-ACUucc pk S3-cuug pk S3-2bp-cuug pk 1.00E+01 2.5 logs at 5 h pi and by 3.5 logs at 8 h pi, while the growth kinetics for S3L2-ACUucc, a mutant that does not form dimers, lagged behind at $1 log increase at 5 h pi and $2.5 logs by 8 h pi ( Figure 8B ). Our analysis demonstrates that disruption of the palindrome via silent codon changes does impede growth kinetics for S3L2-ACUucc pk at early times pi; however, by 12 h pi, similar titers were detected (data not shown).