Frameshifting efficiency.

<p><b>(A)</b> Schematic of the MHV frameshifting signal comprising a slippery heptanucleotide, U_UUA_AAC, and downstream pseudoknot stimulatory structure. <b>(B)</b> Frameshifting efficiencies estimated from the ratio of RiboSeq density in ORF1b to that in ORF1a (red). For comparison, the same calculation was done for RNASeq (green). ORF1a and ORF1b are both present only on the genomic RNA so the ratio of RNASeq densities in the two ORFs is expected to approximate unity. <b>(C)</b> Frameshifting efficiencies for MHV, IBV and HIV-1 frameshift cassettes determined using dual luciferase assays in 17 Cl-1 and BHK-21 cells. Cells were transfected with pDLuc-MHV, pDLuc-IBV or pDLuc-HXB2, and 24 h later, lysates were prepared and assayed for <i>Renilla</i> and firefly luciferase activity.</p

Translation of the genomic RNA uORF.

<p><b>(A)</b> RiboSeq (CHX and HAR) and RNASeq (RNA) counts are shown for repeat 1 at 5 h p.i.; RiboSeq HAR counts are also shown for the high MOI infection. Histograms show the positions of the 5′ ends of reads with a +12 nt offset to map the approximate P-site. Reads whose 5′ ends map to the first, second or third positions of codons relative to the reading frames of the uORFs and ORF1a (which are in phase) are indicated in purple, blue or orange, respectively. Note that the illustrated region does not extend to the genomic 5′ terminus. <b>(B)</b> Comparison of RiboSeq CHX densities summed over all host NCBI RefSeq mRNAs and summed over mRNAs whose annotated coding sequences begin with AUG-CCN (Met-Pro). Histograms show the positions of the 5′ ends of reads, e.g. RPFs of ribosomes paused during initiation (AUG in the P-site at position 0 to 2) have 5′ ends that map predominantly to −12 or −13.</p

RNA synthesis and translation in the 5′ UTR and 3′ ORF regions.

<p>RiboSeq HAR (dark red), RiboSeq CHX (red) and RNASeq (green) densities at 5 h p.i. (repeat 1) in reads per million mapped reads (RPM), smoothed with a 15-nt running mean filter and plotted on a linear scale. Histograms show the positions of the 5′ ends of reads with a +12 nt offset to map (for RPFs) approximate P-site positions. Negative-sense reads are shown in dark blue below the horizontal axis.</p

Time course of MHV total RNA synthesis and translation.

<p><b>(A)</b> Time course of total virus RNA accumulation (left) and total virus translation (right). To normalize for differing library sizes, read counts are expressed relative to the total number of mapped virus RNA (positive and negative-sense) and mapped host mRNA reads for the library. Grey symbols with downward pointing arrows correspond to contaminated samples (see text) and represent upper bounds on the virus fraction. <b>(B)</b> Similar data represented on a linear scale; hatched bars—repeat 1, solid bars—repeat 2. <b>(C)</b> 17Cl-1 cells were infected with MHV-A59 (MOI 10) and harvested at 1, 2.5, 5 and 8 h p.i. Cell lysates were separated by 10% (for N and S westerns) or 17% (for nsp9 western) SDS-PAGE and immunoblotted using monoclonal anti-N, anti-S and anti-nsp9 sera. Molecular masses (in kDa) are indicated on the left. GAPDH was used as a loading control. All viral proteins were detected with a green fluorescent secondary antibody, and GAPDH with a red fluorescent secondary antibody.</p

Comparison of host and virus translation efficiencies.

<p>The translation efficiencies of virus mRNAs were calculated as described in the caption to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005473#ppat.1005473.g004" target="_blank">Fig 4</a>. Host mRNA translation efficiencies are based on the ratio (after normalization for library size) of all RiboSeq or RNASeq reads mapping to any annotated coding region of any splice form of a given gene (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005473#sec019" target="_blank">Methods</a>). Host data are shown only for genes with >100 mapped RNASeq coding-region reads (prior to normalization for library size). Horizontal dashed lines indicate the mean values for host cell genes.</p

Analysis of translation upstream of other annotated ORFs.

<p>RiboSeq (CHX and HAR) and RNASeq (RNA) counts are shown for repeat 1 at 5 h p.i. Histograms show the positions of the 5′ ends of reads with a +12 nt offset to map the approximate P-site. Reads whose 5′ ends map to the first, second or third positions of codons relative to the reading frame of the main annotated ORF (i.e. HE, 4b or 5, respectively) are indicated in purple, blue or orange, respectively. <b>(A)</b> 5′ of the HE ORF. A defective TRS for a very low abundance HE mRNA is annotated with an open green box. In MHV-A59, the HE ORF is disrupted with a premature termination codon (red diamond). Out-of-frame AUG codons that would inhibit ribosomal access via leaky scanning to the next HE-frame AUG codon downstream of the premature termination codon are indicated in green. <b>(B)</b> 5′ of ORF4. In MHV-A59, ORF4 is split by a frameshift mutation into ORF 4b (grey) and a very short ORF4a (pale yellow). An upstream AUU-initiated short ORF and a short out-of-frame AUG-initiated ORF are shown in orange. <b>(C)</b> 5′ of ORF5. A CUG codon in the same frame as the upstream ORF4, and a short out-of-frame AUG-initiated ORF are indicated.</p

Comparison of estimators of relative mRNA abundance.

<p>Relative abundances of the different mRNA species (positive-sense) at 5 h p.i. were estimated either from mean RNASeq density (decumulated as described in the caption to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005473#ppat.1005473.g004" target="_blank">Fig 4</a>) or from the abundance of leader/body “chimeric” RNASeq reads spanning the corresponding TRS junction site. RNASeq densities are expressed in reads per kb per million mapped reads (RPKM). Chimeric TRS read counts are expressed in reads per million mapped reads (RPM).</p

Ribosome pause sites in ORF1a.

<p><b>(A)</b> Histograms of log fold-change from the mean in ORF1a (5 h p.i., repeat 1) showing that RiboSeq densities are more variable than RNASeq densities. RNASeq and RiboSeq counts in ORF1a were first smoothed with a 3-nt running mean filter to average out the intra-codon variability (i.e. triplet periodicity) present in RiboSeq data. <b>(B)</b> Blue triangles indicate selected sites of RPF accumulation in ORF1a, indicative of ribosomal pausing (see text). Histograms show the positions of the 5′ ends of reads with a +12 nt offset to map the approximate P-site. RPF distributions were smoothed with a 15-nt running-mean filter (which, incidentally, reduces the peak height ~15-fold, cf. part C). <b>(C)</b> Enlarged view of the two pause sites without smoothing. The 3′ pause corresponds to reads with 5′ ends mapping to genomic coordinate 11366 while the positions of the 5′ ends of reads at the 5′ pause site differ by 5 nt between the two repeats (genomic coordinate 4704 and 4699, respectively). Reads whose 5′ ends map to the first, second or third positions of codons are indicated in purple, blue or orange, respectively.</p

Translation of the N and I proteins.

<p><b>(A)</b> RiboSeq (CHX and HAR) and RNASeq (RNA) counts are shown for repeat 1 at 5 h p.i. Histograms show the positions of the 5′ ends of reads with a +12 nt offset to map the approximate P-site. Reads whose 5′ ends map to the first, second or third positions of codons relative to the reading frames of the N ORF are indicated in purple, blue or orange, respectively. The I ORF is in the +1 reading frame relative to the N ORF. <b>(B)</b> I is expressed in infected-cells. 17 Cl-1 cells were infected with MHV-A59 and harvested at 1, 2.5, 5 and 8 h p.i. Cell lysates were separated on a 12% SDS-PAGE gel and immunoblotted using monoclonal anti-N and polyclonal anti-I sera. Protein molecular weight markers (MW, kDa) are indicated on the left. N and I were detected with green and red fluorescent secondary antibodies, respectively. <b>(C)</b> Time course of translation of pcDNA.3 N-ORF-derived mRNA in RRL. Translation was at 26°C and samples were collected at the indicated times prior to separation on a 10% SDS-PAGE gel. Labelled polypeptides were detected by autoradiography. Products migrating at the expected sizes for N (50 kDa) and I (23 kDa) are indicated. <b>(D)</b> The pcDNA.3 N-ORF-derived mRNA was translated in RRL and immunoprecipitated with specific anti-N, anti-I or anti-S sera. In the H₂O control, water replaces mRNA template. Immunoprecipitated products were separated on a 10% SDS-PAGE gel and detected as above. <b>(E)</b> Top: Phasing of RPFs (CHX, 5 h p.i.) mapping to the region of the N ORF that is overlapped by the I ORF and the region of the N ORF downstream of the I ORF. Bottom: Phasing as a function of position within the N ORF smoothed with a 55-codon running-mean filter. The bar indicates 55 codons length.</p

MHV RNA synthesis and translation.

<p><b>(A)</b> Transcript map of the 31335-nt MHV-A59 genome. Polyproteins pp1a and pp1b are translated from the genomic RNA, with pp1b being expressed as a transframe fusion with pp1a (i.e. pp1ab) via −1 programmed ribosomal frameshifting (−1 PRF). The 3′ ORFs are expressed from a series of subgenomic RNAs produced during infection. Each subgenomic RNA contains a 5′ leader sequence that is identical to the 5′ leader of the genome, appended via polymerase “jumping” between body transcription regulatory sequences (TRSs) (green diamonds) and the leader TRS (orange diamond) during negative-strand synthesis. Due to mutations present in this laboratory-adapted strain, the hemagglutinin-esterase and ORF4 gene fragments (HE and 4; grey boxes) are not expected to be translated. <b>(B)</b> RiboSeq CHX (red) and RNASeq (green) densities at 5 h p.i. (repeat 1) in reads per million mapped reads (RPM). Read densities are plotted on a log(1+x) scale to cover the wide range in expression across the genome. Histograms show the positions of the 5′ ends of reads with a +12 nt offset to map (for RPFs) approximate P-site positions. Negative-sense reads are shown in dark blue below the horizontal axis. <b>(C)</b> The positive-sense RiboSeq/RNASeq ratio, after first applying a 15-nt running mean (RM) filter to each individual distribution.</p