Nsp1 promotes translation initiation on CoV2 gRNA.

(a) Line plots of individual viral proteins quantified by MS in each fraction. Each line represents a single replicate. (b) Vero cells were transfected with plasmids encoding for individual CoV2 proteins. At 48 hours post-transfection, cells were transfected with nLuc mRNA flanked by either CoV2 (orange) or GAPDH (white) UTRs. At 4 hours post-second transfection, cells were subjected to either luminescence measurements or sucrose gradients coupled to qPCR of nLuc mRNA. (c) nLuc luminescence. Shown are means±SD of 3 independent replicates. (d) Cells transfected, as above, with either GFP or Nsp1 followed by GAPDH-nLuc or CoV2-nLuc mRNA, were lysed and fractionated on 10–50% sucrose gradients with continuous monitoring of rRNA. The content of nLuc mRNA in each fraction was determined by qPCR. Left, rRNA absorbance profiles. Right, percent of GAPDH-nLuc or CoV2-nLuc mRNA found in polysome fractions of sucrose gradients. Shown are qPCR measurements of 4 polysome fractions from 2 independent gradients. P, two-tailed Student’s t-test p-value.