The family Potyviridae includes >30% of known plant virus species, many of which are of great agricultural significance. These viruses have a positive sense RNA genome that is Ϸ10 kb long and contains a single long ORF. The ORF is translated into a large polyprotein, which is cleaved into Ϸ10 mature proteins. We report the discovery of a short ORF embedded within the P3 cistron of the polyprotein but translated in the ؉2 reading-frame. The ORF, termed pipo, is conserved and has a strong bioinformatic coding signature throughout the large and diverse Potyviridae family. Mutations that knock out expression of the PIPO protein in Turnip mosaic potyvirus but leave the polyprotein amino acid sequence unaltered are lethal to the virus. Immunoblotting with antisera raised against two nonoverlapping 14-aa antigens, derived from the PIPO amino acid sequence, reveals the expression of an Ϸ25-kDa PIPO fusion product in planta. This is consistent with expression of PIPO as a P3-PIPO fusion product via ribosomal frameshifting or transcriptional slippage at a highly conserved G 1-2A6-7 motif at the 5 end of pipo. This discovery suggests that other short overlapping genes may remain hidden even in well studied virus genomes (as well as cellular organisms) and demonstrates the utility of the software package MLOGD as a tool for identifying such genes.P3 ͉ PIPO ͉ Potyvirus ͉ Turnip mosaic virus ͉ frameshift

BEL1-like transcription factors interact with Knotted1 types to regulate numerous developmental processes. In potato (Solanum tuberosum), the BEL1 transcription factor St BEL5 and its protein partner POTH1 regulate tuber formation by mediating hormone levels in the stolon tip. The accumulation of St BEL5 RNA increases in response to short-day photoperiods, inductive for tuber formation. RNA detection methods and heterografting experiments demonstrate that BEL5 transcripts are present in phloem cells and move across a graft union to localize in stolon tips, the site of tuber induction. This movement of RNA to stolon tips is correlated with enhanced tuber production. Overexpression of BEL5 transcripts that include the untranslated sequences of the BEL5 transcript endows transgenic lines with the capacity to overcome the inhibitory effects of long days on tuber formation. Addition of the untranslated regions leads to preferential accumulation of the BEL5 RNA in stolon tips under short-day conditions. Using a leaf-specific promoter, the movement of BEL5 RNA to stolon tips was facilitated by a short-day photoperiod, and this movement was correlated with enhanced tuber production. These results implicate the transcripts of St BEL5 in a long-distance signaling pathway that are delivered to the target organ via the phloem stream.

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.

Translationally competent mRNAs form a closed loop via interaction of initiation factors with the 5' cap and poly(A) tail. However, many viral mRNAs lack a cap and/or a poly(A) tail. We show that an uncapped, nonpolyadenylated plant viral mRNA forms a closed loop by direct base-pairing (kissing) of a stem loop in the 3' untranslated region (UTR) with a stem loop in the 5' UTR. This allows a sequence in the 3' UTR to confer translation initiation at the 5'-proximal AUG. This base-pairing is also required for replication. Unlike other cap-independent translation mechanisms, the ribosome enters at the 5' end of the mRNA. This remarkably long-distance base-pairing reveals a novel mechanism of cap-independent translation and means by which mRNA UTRs can communicate.

For recognition by the translational machinery, most eukaryotic cellular mRNAs have a 5' cap structure [e.g. m7G(5')ppp(5')N]. We describe a translation enhancer sequence (3'TE) located in the 3'-untranslated region (UTR) of the genome of the PAV barley yellow dwarf virus (BYDV-PAV) which stimulates translation from uncapped mRNA by 30- to 100-fold in vitro and in vivo to a level equal to that of efficient capped mRNAs. A four base duplication within the 3'TE destroyed the stimulatory activity. Efficient translation was recovered by addition of a 5' cap to this mRNA. Translation of both uncapped mRNA containing the 3'TE in cis and capped mRNA lacking any BYDV-PAV sequence was inhibited specifically by added 3'TE RNA in trans. This inhibition was reversed by adding initiation factor 4F (eIF4F), suggesting that the 3'TE, like the 5' cap, mediates eIF4F-dependent translation initiation. The BYDV-PAV 5'UTR was necessary for the 3'TE to function, except when the 3'TE itself was moved to the 5'UTR. Thus, the 3'TE is sufficient for recruiting the translation factors and ribosomes, while the viral 5'UTR may serve only for the long distance 3'-5' communication. Models are proposed to explain this novel mechanism of cap-independent translation initiation facilitated by the 3'UTR.