Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement

Abstract

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5’ end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.

Synergistic infection of BrYV and PEMV 2 increases the accumulations of both BrYV and BrYV-derived siRNAs in Nicotiana benthamiana

“…It has been reported that polerovirus PLRV contains three subgenomic RNAs (sgRNAs) at 3′-region of viral RNA5152. Translation of sgRNA1 enables expression of ORFs 3a, 3, 4, and 54153, while that of sgRNA2 codes two proteins at the 3′-region of ORF551. The recently identified sgRNA3 encodes a RNA-binding protein52.…”

Defining Viral Defective Ribosomal Products: Standard and Alternative Translation Initiation Events Generate a Common Peptide from Influenza A Virus M2 and M1 mRNAs

“…Translation initiation at Leu 59 would create a polypeptide of 40 amino acids, which would make it the shortest described IAV polypeptide, but still possibly functional. M42, a splice variant of M2, is 54 residues in length, (48), and a functional 45-residue protein has been defined in Luteroviradae (49). Our findings imply that translation can be also be initiated at Leu 96, which would create a 2 amino acid gene product before the stop codon at position 98--surely too short to be functional.…”

Cis- and trans-regulation of luteovirus gene expression by the 3′ end of the viral genome

“…1). All luteoviruses and poleroviruses contain a 45 – 50 codon ORF (ORF3a) beginning with a non-AUG codon (AUU, AUA, ACG, or CUG depending on the virus) and terminating between the start codons of ORFs 3 (CP ORF) and ORF 4 (Smirnova et al, 2015). [WAM7] Owing to the lack of an AUG codon, ORF 3a is translated at low levels, so most ribosomes (40S subunits) do not initiate at the ORF3a start codon and continue to scan until they initiate translation at the ORF 3 (CP) or ORF4 start codon.…”