Fig. 1.

MuLV RNA synthesis and translation. a Map of the Moloney MuLV proviral DNA, showing the locations of the untranslated 3′ (U3), R, and U5 repeats within the LTRs, and the gag, pol and env coding regions. U3 houses the viral promoter region, while the transcription start site (TSS) corresponds to the first nucleotide of the R region. The poly-adenylation (poly-A) signal also occurs within R. Translation of pol is dependent upon readthrough of the gag gene termination codon. Two distinct mRNA species are produced from the provirus template, with the gag-pol transcript being unspliced and the env transcript being spliced at the indicated positions. b Rat2 cells were infected with MuLV and at 96 h p.i. cell lysates were prepared, separated by 10% SDS-PAGE and immunoblotted using a polyclonal anti-capsid (p30) serum. Molecular weights (MW; in kDa) are indicated on the left. GAPDH was used as a loading control. Viral proteins were detected with a green fluorescent secondary antibody, and GAPDH was detected with a red fluorescent secondary antibody. c RiboSeq CHX (red) and RNASeq (purple) densities in reads per million mapped reads (RPM), smoothed with a 31-nt sliding window. Negative-sense reads are shown in dark blue below the horizontal axes. d Bar plot showing the density of RPFs mapped to the pol gene relative to the gag gene, normalised by the equivalent density of RNASeq reads in these regions. The ratio of pol to gag translation is approximately 0.07, on average; indicating that ~ 7% of ribosomes undergo stop codon readthrough to translate pol