The Multifunctional Long-Distance Movement Protein of Pea Enation Mosaic Virus 2 Protects Viral and Host Transcripts from Nonsense-Mediated Decay

Abstract

The nonsense-mediated decay (NMD) pathway presents a challenge for RNA viruses with termination codons that precede extended 3= untranslated regions (UTRs). The umbravirus Pea enation mosaic virus 2 (PEMV2) is a nonsegmented, positive-sense RNA virus with an unusually long 3= UTR that is susceptible to NMD. To establish a systemic infection, the PEMV2 long-distance movement protein p26 was previously shown to both stabilize viral RNAs and bind them for transport through the plant's vascular system. The current study demonstrated that p26 protects both viral and nonviral messenger RNAs from NMD. Although p26 localizes to both the cytoplasm and nucleolus, p26 exerts its anti-NMD effects exclusively in the cytoplasm independently of long-distance movement. Using a transcriptome-wide approach in the model plant Nicotiana benthamiana, p26 protected a subset of cellular NMD target transcripts, particularly those containing long, structured, GC-rich 3= UTRs. Furthermore, transcriptome sequencing (RNA-seq) revealed that the NMD pathway is highly dysfunctional during PEMV2 infection, with 1,820 (48%) of NMD targets increasing in abundance. Widespread changes in the host transcriptome are common during plant RNA virus infections, and these results suggest that, in at least some instances, virus-mediated NMD inhibition may be a major contributing factor. IMPORTANCE Nonsense-mediated decay (NMD) represents an RNA regulatory pathway that degrades both natural and faulty messenger RNAs with long 3= untranslated regions. NMD targets diverse families of RNA viruses, requiring that viruses counteract the NMD pathway for successful amplification in host cells. A protein required for long-distance movement of Pea enation mosaic virus 2 (PEMV2) is shown to also protect both viral and host mRNAs from NMD. RNA-seq analyses of the Nicotiana benthamiana transcriptome revealed that PEMV2 infection significantly impairs the host NMD pathway. RNA viruses routinely induce large-scale changes in host gene expression, and, like PEMV2, may use NMD inhibition to alter the host transcriptome in an effort to increase virus amplification.

Structural Analysis and Whole Genome Mapping of a New Type of Plant Virus Subviral RNA: Umbravirus-Like Associated RNAs

“…Arabidopsis thaliana protoplasts were inoculated with in vitro synthesized CYVaV transcripts containing the exact 5′ end and one non-template residue at the 3′ end, and total RNA was isolated 24 h later. Since efficient accumulation of PEMV2 in protoplasts is dependent on the presence of the ORF3 product p26 [ 11 , 32 ], which is absent in CYVaV, cells were also inoculated separately or together with PEMV2 RNA, and probes complementary to CYVaV and PEMV2 plus (+)strands were combined and used for Northern blot analysis. As a control, CYVaV transcripts containing mutations in sequence required for frame-shifting (CYVaV-fsm; shown in Figure 8) were also assayed for accumulation.…”

“…Since the sequences of these two hairpins share no extensive similarity among Class 2 ulaRNAs ( Figure 10 C), conservation of a two hairpin structure at this location may be necessary for some function. From their location in the vicinity of the RdRp termination codon, one possibility is that one or both of these hairpins are involved in protection against NMD, as NMD-protective sequences, if present, are located just downstream of a termination codon [ 11 , 67 , 68 ]. NMD is a eukaryotic mRNA surveillance pathway that detects and eliminates mRNAs containing premature termination codons, leading to lengthy 3′ UTRs.…”

“…All mutations were verified by sequencing. pCB-PEMV2 (pCB301 harboring full-length PEMV2 gRNA) was previously described [ 11 ]. GFP-3′UTR PEMV (the 3′ UTR of PEMV2 immediately downstream of the GFP ORF in pBIN61S-GFP) was also previously described [ 11 , 25 ].…”

“…pCB-PEMV2 (pCB301 harboring full-length PEMV2 gRNA) was previously described [ 11 ]. GFP-3′UTR PEMV (the 3′ UTR of PEMV2 immediately downstream of the GFP ORF in pBIN61S-GFP) was also previously described [ 11 , 25 ]. GFP-3′UTR CYVaV was constructed by ligating a DNA fragment encompassing the CYVaV 3′ UTR into the BamHI and XbaI restriction sites of pBIN61S-GFP.…”

“…The mutualistic association between umbraviruses and their helper viruses is demonstrated by helper virus acquisition of cell-to-cell movement through shared use of the ORF4 umbravirus product, and umbraviruses gaining a suppressor of RNA silencing and genome encapsidation from their associated virus. In addition, since the umbravirus ORF3 product is multifunctional and also responsible for protecting the umbravirus sgRNA (and many cellular mRNAs) from nonsense mediated decay (NMD) [ 11 ], the helper virus likely benefits from this protection as well.…”