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

Abstract

Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they 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 (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 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 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of 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 distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2'-hydroxyl acylation and primer extension (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 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. Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in the by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains in (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strat...

UGA stop codon readthrough to translate intergenic region of Plautia stali intestine virus does not require RNA structures forming internal ribosomal entry site

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“…After this paper was submitted, investigations into the AMD1 gene and all human genes containing UGA_CUAG were reported in Yordanova et al (2018) and Loughran et al (2018), respectively. Also, the long-range RNA interaction of another member of luteovirus was reported in Xu et al (2018).…”

Introduction of a leaky stop codon as molecular tool in Chlamydomonas reinhardtii

“…Like all biological processes, translation termination is associated with a certain error rate, which is not identical between the three stop codons in the standard code. This error rate can further be influenced by RNA secondary structures, or simply the immediate sequence environment of the stop codon, meaning that certain sequence constellations can lead to significant read-through rates on the order of 1–10% [ 34 , 48 – 50 ]. In eukaryotes, leaky stop codons appear to be generally selected against [ 51 , 52 ], although examples where read-through fulfills a regulatory function have been documented [ 53 , 54 ].…”

The Interaction Dynamics of Two Potato Leafroll Virus Movement Proteins Affects Their Localization to the Outer Membranes of Mitochondria and Plastids

“…Mutants tested included the ΔCP mutant that only expresses the non-incorporated form of the RTP due to a deletion of the leaky stop codon between ORF3 and ORF5 [ 10 ], a P17-null mutant (ΔP17) that does not express the movement protein [ 18 ], the ΔRTD mutant, which expresses CP but lacks the RTD [ 15 , 16 , 28 , 29 ], and a ΔRTC mutant (referred to as RTC-3 in Reference [ 30 ]) where the last 218 C-terminal residues of the RTD are truncated. All mutants except ΔCP can assemble virions and all exhibit varying degrees of host-dependent delays in systemic movement [ 15 , 16 , 18 , 30 ]. Mock infiltrated tissue was used as a negative control and three biological replicate APs with two MS injection replicates were analyzed [ 31 ] for each PLRV infection condition.…”