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 if the differences in viral growth kinetics were due to reduced levels of RNA replication and/or synthesis, we harvested total RNA at 0, 5, 8 and 12 h pi and performed northern blot analysis. Total RNA was probed using a nucleocapsid specific biotinylated probe ( Figure 9A ) to examine levels of gRNA. S3L2-ACUucc gRNA levels were reduced $2-fold when compared with SARS-CoV at 5 and 8 h pi, suggesting that RNA replication contributed to the reduction in viral titers ( Figure 9B ). To determine the quantities of sgRNA species, the total RNA was enriched for poly A containing mRNA species ( Figure 9C ) before separation on agarose gel. All sgRNA species were readily detectible in both SARS-CoV and S3L2-ACUucc samples; however, $1.5-fold reduction in the quantity of sgRNA species was detected at 5 and 8 h pi ( Figure 9D ), suggesting that reduced levels of RNA transcription also contributed to the titer loss. Finally we compared the levels of ORF1a and ORF1b replicase proteins to determine if these protein levels were altered as well. Equivalent protein concentrations were probed with polyclonal rabbit antisera directed against nsp1 or nsp16 as indicated ( Figure 9E and F). Reduced ORF1a and ORF1b replicase protein levels were seen at 5 and 8 h pi but similar levels were detected by 12 h pi (Supplementary Figure S5 ). Together these data indicate that dimerization is not required for viral viability but it is important for maintaining efficient rates of virus growth and genomic and subgenomic mRNA synthesis.