TRS1 rpS6 28S 18S B A Oligo(dT) Input +MN TRS1 +m 7 GTP His PABP Figure S1. pTRS1 associates with the mRNA body independent of the mRNA cap and co- sediments with 40S ribosomal subunits. (A) Cells were transfected as in Fig. 1C, and lysates were incubated with oligo (dT) cellulose in the presence or absence of MN or free m 7 GTP. (B) BADinGFP infected cells (72 hpi) were treated with puromycin (1 µM) for 1 hour before harvest to disrupt ribosomes. Cytoplasmic lysates were resolved on 5-20% sucrose gradients, and the presence of pTRS1 or the ribosomal protein rpS6 in each fraction was determined by Western blot (top panel). Ribosomal RNAs (28S, 18S; bottom panel) in each fraction were visualized on agarose gels. Fig. S1

FL UL99 5’UTR trunc UL99 5’UTR pGL3 control RNA TRS1 Expression Vector (μg) A Figure S2. pTRS1 stimulates reporter expression in a dose dependent manner. (A) Increasing amounts of pTRS1 expression vector were co-transfected with a constant amount of pGL3- Control luciferase plasmid containing either the control 5’UTR, UL99 5’UTR or truncated UL99 5’UTR. The graph shows the fold change in luciferase activity (open boxes) and luciferase RNA abundance (closed boxes) compared to samples transfected with a control vector expressing GFP. (n=8) (B) Increasing concentrations of pTRS1 expression vector were transfected as in (A) and pTRS1 abundance was determined by Western blot. pTRS1 Tubulin B Fig. S2

Figure S3. pTRS1 binds the m 7 G mRNA cap independent of the PKR and RNA binding domains. (A) GFP, wild type pTRS1 or pTRS1 PKR binding domain mutant expression vectors were transfected and analyzed as in Fig. 1C. (B) Wild type or pTRS1 RNA binding domain mutant expression vectors were transfected and analyzed as in Fig. 1C. A GFP TRS1-WT TRS1-dPBD IP m 7 G Input IP m 7 G Input TRS1- dRBD TRS1-WT B Fig. S3

Figure S4. pTRS1 stimulates the rate of translation. (A) GFP or pTRS1 expression vectors were transfected and metabolically labelled as in Fig. 3B. At the indicated times the amount of radiolabel incorporated was measured as in Fig. 3B. The rate of incorporation was plotted as the fold change relative GFP transfected cells after 5 minutes of labelling. (n=3) GFP TRS1 TRS1 + CHX A

INElution Sepharose M 7 G-Sepharose Figure S5. Proteins were captured with either Sepharose beads or Sepharose beads with m 7 G bound. Bound proteins were eluted with excess free m 7 G. Eluted proteins were visualized by Coomassie staining