A stem-loop structure in Potato leafroll virus open reading frame 5 (ORF5) is essential for readthrough translation of the coat protein ORF stop codon 700 bases upstream

Biblio

Publication Type:

Journal Article

Source:

J. Virol., Volume 92 (2018)

URL:

http://jvi.asm.org/content/92/11/e01544-17.full

Abstract:

<p>Translational readthrough of the stop codon of the capsid protein (CP)<br />
open reading frame (ORF) is used by members of the Luteoviridae to produce their<br />
minor capsid protein as a readthrough protein (RTP). The elements regulating RTP<br />
expression are not well understood, but they involve long-distance interactions between<br />
RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine<br />
were identified as the primary amino acids inserted at the stop codon of<br />
Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidinerich<br />
domain immediately downstream and a branched stem-loop structure 600 to<br />
700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt<br />
and restore the base of the distal stem-loop structure prevented and restored stop<br />
codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted<br />
to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon.<br />
Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf<br />
virus was not compatible with the stop codon-proximal element of PLRV in facilitating<br />
readthrough. Moreover, deletion of the complementary tract of bases from the<br />
stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast,<br />
the distance and sequence composition between the two domains was flexible. Mutants<br />
deficient in RTP translation moved long distances in plants, but fewer infection<br />
foci developed in systemically infected leaves. Selective 2=-hydroxyl acylation and<br />
primer extension (SHAPE) probing to determine the secondary structure of the mutant<br />
DRTEs revealed that the functional mutants were more likely to have bases accessible<br />
for long-distance base pairing than the nonfunctional mutants. This study<br />
reveals a heretofore unknown combination of RNA structure and sequence that reduces<br />
stop codon efficiency, allowing translation of a key viral protein.</p>