Date Published: November 19, 2018
Publisher: Public Library of Science
Author(s): Jared P. May, Xuefeng Yuan, Erika Sawicki, Anne E. Simon, Aiming Wang.
Nonsense-mediated decay (NMD) is a host RNA control pathway that removes aberrant transcripts with long 3’ untranslated regions (UTRs) due to premature termination codons (PTCs) that arise through mutation or defective splicing. To maximize coding potential, RNA viruses often contain internally located stop codons that should also be prime targets for NMD. Using an agroinfiltration-based NMD assay in Nicotiana benthamiana, we identified two segments conferring NMD-resistance in the carmovirus Turnip crinkle virus (TCV) genome. The ribosome readthrough structure just downstream of the TCV p28 termination codon stabilized an NMD-sensitive reporter as did a frameshifting element from umbravirus Pea enation mosaic virus. In addition, a 51-nt unstructured region (USR) at the beginning of the TCV 3’ UTR increased NMD-resistance 3-fold when inserted into an unrelated NMD-sensitive 3’ UTR. Several additional carmovirus 3’ UTRs also conferred varying levels of NMD resistance depending on the construct despite no sequence similarity in the analogous region. Instead, these regions displayed a marked lack of RNA structure immediately following the NMD-targeted stop codon. NMD-resistance was only slightly reduced by conversion of 19 pyrimidines in the USR to purines, but resistance was abolished when a 2-nt mutation was introduced downstream of the USR that substantially increased the secondary structure in the USR through formation of a stable hairpin. The same 2-nt mutation also enhanced the NMD susceptibility of a subgenomic RNA expressed independently of the genomic RNA. The conserved lack of RNA structure among most carmoviruses at the 5’ end of their 3’ UTR could serve to enhance subgenomic RNA stability, which would increase expression of the encoded capsid protein that also functions as the RNA silencing suppressor. These results demonstrate that the TCV genome has features that are inherently NMD-resistant and these strategies could be widespread among RNA viruses and NMD-resistant host mRNAs with long 3’ UTRs.
Since viruses are obligate intracellular parasites, host cellular RNA control pathways must be tolerated or circumvented for successful virus amplification. One such pathway is nonsense-mediated decay (NMD), which normally removes aberrant mRNAs containing premature termination codons (PTCs) to prevent detrimental effects caused by the expression of truncated proteins. The mechanism of NMD has been extensively studied for PTC-containing mRNAs with exon-junction complexes (EJCs) deposited during splicing downstream of the PTC (see  for a review). The EJC is associated with NMD factors and is removed by the ribosome-associated PYM protein during normal translation, but remains unaltered in PTC-containing mRNAs, which leads to NMD . Key NMD factors that are conserved across nearly all eukaryotes  include the Up-frameshift group of proteins (UPF1-3), with UPF1 as the master regulator . UPF1 contains ATPase and RNA helicase domains necessary for NMD and is regulated by a complex involving UPF2 and UPF3 [5, 6]. Activated UPF1 recruits nucleases to eliminate NMD-targeted transcripts, with key differences existing between mammalian and plant systems . For example, both the SMG6 endo- and SMG7-guided exonucleolytic pathways are utilized in mammalian cells [8, 9], whereas only SMG7-coordinated exonucleolytic cleavage is used in plants due to the absence of SMG6 homologs [10, 11].
Multiple studies have established that diverse families of viruses are targeted by NMD [37, 54]. However, given that most published NMD-evasion mechanisms are virus-specific [27, 31–33], it remains unclear if general mechanisms exist, aside from PTBP1 binding , that are shared with cellular mRNAs whose otherwise extensive 3’ UTRs would comprise natural targets. For the first time, we have identified features of a virus genome that provide NMD-resistance without sequence specificity, which could be employed across diverse families of viruses and by host mRNAs.