The transcriptional and translational landscape of equine torovirus

Hazel Stewart; Katherine Brown; Adam M. Dinan; Nerea Irigoyen; Eric J. Snijder; Andrew E. Firth

Materials and Methods) to account for the fact that not all of the RNA-seq density in 163 the 3' ORFs derives from transcripts from which the ORFs can be expressed. Ribo-seq 164 coverage is much higher towards the 3' end of the genome, particularly across the M 165 and N genes, reflecting the translation of abundant subgenomic RNAs in this region 166 ( Figure 2, Figure 3 ). ORFs 1a and 1b contain a considerably lower density of Ribo-seq 167 reads. The relatively low translation efficiencies calculated for ORFs 1a and 1b may 168 be partly due to some gRNA being packaged (or destined for packaging) and 169 unavailable for translation but still contributing to the estimate of gRNA RNA-seq 170 density. ORF1a has a higher Ribo-seq density and a higher translational efficiency 171 than ORF1b, reflecting the proportion of ribosomes terminating at the ORF1a stop 172 codon and not undergoing the -1 frameshift into ORF1b (Figure 2, Figure 3 ). As 173 expected, RNA-seq density is similar across ORF1a and ORF1b, as both are present 174 only on the full-length genomic RNA ( Figure 2 ). The region covering the HE ORF also 175 has low ribosomal coverage (Figure 2 ), which may be due to the fact that the EToV 176 HE gene is nonfunctional due to a large deletion including the canonical AUG (16). HE 177 is not shown in Figure 3 as the HE transcript is much less abundant than the 178 "upstream" M transcript which makes the decumulation procedure susceptible to 179 noise (see Irigoyen et al., 2016) . Translational efficiency appears highest for the M 180 and S subgenomic RNAs. The high RNA-seq density in the 5' UTR may be indicative of 181 one or more defective interfering (DI) RNAs in the sample (see below). Ribosome 182 protected fragments (RPFs) were also identified mapping to the second half of the 5' 183 UTR, mostly in the +2/-1 frame with respect to ORF1a ( Figure 2A) . 184