A stem-loop structure in potato leafroll virus ORF5 that is essential for readthrough translation of the coat protein ORF stop codon 700 bases upstream

Authors: XU, YI - Cornell University; JU, HO JUNG - Cornell University; Deblasio, Stacy; CARINO, ELIZABETH - Iowa State University; JOHNSON, R. - University Of Washington; MACCOSS, M. - University Of Washington; Heck, Michelle; MILLER, W. ALLEN - Iowa State University; Gray, Stewart

Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/7/2018
Publication Date: 6/1/2018
Citation: Xu, Y., Ju, H., DeBlasio, S.L., Carino, E.J., Johnson, R., MacCoss, M., Heck, M.L., Miller, W., Gray, S.M. 2018. A stem-loop structure in potato leafroll virus ORF5 that is essential for readthrough translation of the coat protein ORF stop codon 700 bases upstream. Journal of Virology. https://doi.org/10.1128/JVI.01544-17.
DOI: https://doi.org/10.1128/JVI.01544-17

Interpretive Summary: Potato leafroll virus (PLRV) and its relatives are responsible for economically significant disease in potato and other staple food crops. There is limited effective resistance in crops affected by these viruses. Management is limited to insecticidal control of vectors that is only partially effective. This study was designed to investigate how the virus regulates the expression of its proteins that are linked to its ability to move within and between hosts. Understanding these mechanisms identifies potential targets for controlling these viruses. We found protein expression is controlled by the interaction of two areas of the virus genome that are separated by the stretch of gene coding region representing more than 10% of the length of the total virus genome. When the genome folds into a complex 3-dimensional form these two regions are in close proximity and bind together allowing the proteins to be produced. Small changes in either of the two regions can compromise their ability to interact and to produce the correct proteins. The result is a virus that does not move efficiently in its host and therefore not available to be transmitted by its vector to other susceptible hosts. All of the viruses related to PLRV have similar genome structures and function and therefore a single strategy to disrupt the interaction of these regions may provide a common management tool.

Technical Abstract: Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream, and a branched stem-loop structure 600-700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nt of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf virus was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distance in plants, but fewer infection foci developed in systemically infected leaves. SHAPE probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than in the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein.