In the absence of a 5′ cap, plant positive-strand RNA viruses have evolved a number of different elements in their 3′ untranslated region (UTR) to attract initiation factors and/or ribosomes to their templates. These 3′ cap-independent translational enhancers (3′ CITEs) take different forms, such as I-shaped, Y-shaped, T-shaped, or pseudoknotted structures, or radiate multiple helices from a central hub. Common features of most 3′ CITEs include the ability to bind a component of the translation initiation factor eIF4F complex and to engage in an RNA-RNA kissing-loop interaction with a hairpin loop located at the 5′ end of the RNA. The two T-shaped structures can bind to ribosomes and ribosomal subunits, with one structure also able to engage in a simultaneous long-distance RNA-RNA interaction. Several of these 3′ CITEs are interchangeable and there is evidence that natural recombination allows exchange of modular CITE units, which may overcome genetic resistance or extend the virus's host range.

A small open reading frame (ORF), pipo, overlaps with the P3 coding region of the potyviral polyprotein ORF. Previous evidence suggested a requirement for pipo for efficient viral cell-to-cell movement. Here, we provide immunoblotting evidence that the protein PIPO is expressed as a trans-frame protein consisting of the amino-terminal half of P3 fused to PIPO (P3N-PIPO). P3N-PIPO of Turnip mosaic virus (TuMV) fused to GFP facilitates its own cell-to-cell movement. Using a yeast two-hybrid screen, co-immunoprecipitation assays, and bimolecular fluorescence complementation (BiFC) assays, we found that P3N-PIPO interacts with host protein PCaP1, a cation-binding protein that attaches to the plasma membrane via myristoylation. BiFC revealed that it is the PIPO domain of P3N-PIPO that binds PCaP1 and that myristoylation of PCaP1 is unnecessary for interaction with P3N-PIPO. In PCaP1 knockout mutants (pcap1) of Arabidopsis, accumulation of TuMV harboring a GFP gene (TuMV-GFP) was drastically reduced relative to the virus level in wild-type plants, only small localized spots of GFP were visible, and the plants showed few symptoms. In contrast, TuMV-GFP infection in wild-type Arabidopsis yielded large green fluorescent patches, and caused severe stunting. However, viral RNA accumulated to high level in protoplasts from pcap1 plants indicating that PCaP1 is not required for TuMV RNA synthesis. In contrast to TuMV, the tobamovirus Oilseed rape mosaic virus did not require PCaP1 to infect Arabidopsis plants. We conclude that potyviral P3N-PIPO interacts specifically with the host plasma membrane protein PCaP1 to participate in cell-to-cell movement. We speculate that PCaP1 links a complex of viral proteins and genomic RNA to the plasma membrane by binding P3N-PIPO, enabling localization to the plasmodesmata and cell-to-cell movement. The PCaP1 knockout may contribute to a new strategy for recessive resistance to potyviruses. The Potyviridae is the largest and most economically important family of plant viruses. A key step in the life cycle of all plant viruses is transport of the viral genome through the plasmodesmata, highly regulated channels that connect cells. While the mechanisms of cell-to-cell movement of many plant viruses have been characterized, our understanding of Potyviridae movement is lacking. The viral RNA genome is transported to the plasmodesmata by a complex of viral proteins including a recently discovered protein, P3N-PIPO which is encoded in two reading frames. The details of this localization process are unclear. Here, we identify a potential missing link that suggests how the potyviral movement complex may anchor to the plasma membrane including in the plasmodesmata. The host protein PCaP1, a divalent cation-binding plasma membrane protein, binds the P3N-PIPO protein of Turnip mosaic virus (TuMV). Both proteins were detected in the plasma membrane and plasmodesmata. Arabidopsis plants lacking PCaP1 allowed TuMV RNA replication but showed inefficient TuMV movement, reduced TuMV accumulation, and had greatly attenuated symptoms. However, these plants allowed normal infection by a tobamovirus. Thus, mutation of the PCaP1 gene may contribute to breeding potyvirus-resistant crops.

Barley yellow dwarf viruses represent one of the most economically important and ubiquitous groups of plant viruses. This review focuses primarily on four research areas in which progress has been most rapid. These include (a) evidence supporting reclassification of BYDVs into two genera; (b) elucidation of gene function and novel mechanisms controlling gene expression; (c) initial forays into understanding the complex interactions between BYDV virions and their aphid vectors; and (d ) replication of a BYDV satellite RNA. Economic losses, symptomatology, and means of control of BYD are also discussed.

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. In order to maximize coding capacity, RNA viruses often encode overlapping genes and use unusual translational control mechanisms. Plant viruses express proteins required for movement of the virus through the plant, often from non-canonically translated open reading frames (ORFs). Viruses in the economically important Luteoviridae family are confined to the phloem (vascular) tissue, probably due to their specialized phloem-specific movement proteins. These proteins are translated from one viral mRNA, sgRNA1, via initiation at more than one AUG codon to express overlapping genes, and by ribosomal read-through of a stop codon. Here, we describe yet another gene translated from sgRNA1, ORF3a. Translation of ORF3a initiates at a non-standard (not AUG) start codon. 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. P3a localizes in the Golgi apparatus and adjacent to the plasmodesmata, supporting a role in intercellular movement. In summary, we used a powerful bioinformatic tool to discover a cryptic gene whose product is required for movement of a phloem-specific plant virus, revealing multiple levels of translational control that regulate expression of four proteins from a single mRNA.