Figure 1: Alternative reading frame proteins encoded in the P3 coding region of ClYVV.
(a) Schematic diagram of the genomes of two ClYVV variants, Cl30 and RB. The polyprotein expressed from the genome is processed into P1; helper component proteinase (HC-Pro); P3 (green box with vertical stripes); 6K1; cylindrical inclusion (CI); 6K2; nuclear inclusion A (NIa), which is further cleaved into NIa-VPg and proteinase domain (NIa-Pro); nuclear inclusion protein B (NIb); and coat protein (CP). The positions of the pipo ORF (blue box with horizontal stripes), the alt ORF identified in this study (magenta box), and the conserved G1-2A6-7 motif are indicated. (b) Alignment of the nucleotide sequences of part of the pipo ORF of Cl30 and RB. Spaces denote the polyprotein reading frame. The G2A6 motif is underlined. The stop codons of the pipo ORF (–1 stop) and P3N-ALT (+1 stop) are italicized and shaded. The nucleotides are numbered from the start of the P3 ORF. (c) Schematic diagrams of Cl30-P3 and RB-P3 RNAs for in vitro expression analysis, Cl30-P3N-PIPOmk and RB-P3N-PIPOmk RNAs used to prepare size markers for P3N-PIPO, and Cl30-P3N-ALTmk and RB-P3N-ALTmk RNAs used to prepare size markers for P3N-ALT. The mutated nucleotides in G2A6 motif in the RNAs for size marker preparation are indicated by lower case red letters. (d) In vitro translation analysis using WGE. The radiolabeled translation products were visualized by autoradiography. The positions of P3, P3N-PIPO and P3N-ALT are marked on the left side of the panel. Open and closed arrowheads indicate the P3N-PIPO (–1 reading frame) and P3N-ALT (+1 reading frame), respectively. Positions of molecular mass markers (kDa) are indicated on the right side of the panel. A representative image of triplicated experiments is shown.
