Translation of the N and I proteins.
(A) 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) 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. (C) 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. (D) 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. (E) 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.
