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

Ekaterina Smirnova,W Allen Miller,Catherine Reinbold,Betty Y.-W Chung,Véronique Brault,Danièle Scheidecker,Andrew E Firth,Véronique Ziegler-Graff,Aurélie M Rakotondrafara,Shou-Wei Ding

doi:10.1371/journal.ppat.1004868

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.

Highlights

RNA viruses are models of efficiency in compressing maximum information, such as coding and regulatory signals, into minimum sequence space
We found that ORF3a is not required for viral genome replication, but is required for long-distance movement of the virus in the plant
The movement function could be restored in trans by providing the ORF3a product, P3a, from another viral or plasmid vector

Summary

Introduction

RNA viruses are models of efficiency in compressing maximum information, such as coding and regulatory signals, into minimum sequence space. RNA viruses often employ noncanonical translation mechanisms [1, 2]. Many viruses encode genes in overlapping open reading frames (ORFs), some of which can be very short. To decipher a virus life cycle, it is imperative to identify all the coding regions and to understand their function and how they are regulated. Specialized bioinformatic tools are often required to detect key viral genes. We use such tools to identify an essential ORF conserved in the two main genera in the Luteoviridae family, and provide evidence of its role in infection

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