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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.

SUBMITTER: May JP

PROVIDER: S-EPMC7064760 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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

May Jared P JP Johnson Philip Z PZ Ilyas Muhammad M Gao Feng F Simon Anne E AE

mBio 20200310 2

The nonsense-mediated decay (NMD) pathway presents a challenge for RNA viruses with termination codons that precede extended 3' untranslated regions (UTRs). The umbravirus <i>Pea enation mosaic virus 2</i> (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 vascul ...[more]

PMID: 32156817

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Project description:Eukaryotic polycistronic transcription units are rare and only a few examples are known, mostly being the outcome of serendipitous discovery. We claim that nonsense-mediated mRNA decay (NMD) immune structure is a common characteristic of polycistronic transcripts, and that this immunity is an emergent property derived from all functional CDSs. The human RefSeq transcriptome was computationally screened for transcripts capable of eliciting NMD, and which contain an additional ORF(s) potentially capable of rescuing the transcript from NMD. Transcripts were further analyzed implementing domain-based strategies in order to estimate the potential of the candidate ORF to encode a functional protein. Consequently, we predict the existence of forty nine novel polycistronic transcripts. Experimental verification was carried out utilizing two different types of analyses. First, five Gene Expression Omnibus (GEO) datasets from published NMD-inhibition studies were used, aiming to explore whether a given mRNA is indeed insensitive to NMD. All known bicistronic transcripts and eleven out of the twelve predicted genes that were analyzed, displayed NMD insensitivity using various NMD inhibitors. For three genes, a mixed expression pattern was observed presenting both NMD sensitivity and insensitivity in different cell types. Second, we used published global translation initiation sequencing data from HEK293 cells to verify the existence of translation initiation sites in our predicted polycistronic genes. In five of our genes, the predicted rescuing uORFs are indeed identified as translation initiation sites, and in two additional genes, one of two predicted rescuing uORF is verified. These results validate our computational analysis and reinforce the possibility that NMD-immune architecture is a parameter by which polycistronic genes can be identified. Moreover, we present evidence for NMD-mediated regulation controlling the production of one or more proteins encoded in the polycistronic transcript.

Project description:Many plant viruses without 5' caps or 3' poly(A) tails contain 3' proximal, cap-independent translation enhancers (3'CITEs) that bind to ribosomal subunits or translation factors thought to assist in ribosome recruitment. Most 3'CITEs participate in a long-distance kissing-loop interaction with a 5' proximal hairpin to deliver ribosomal subunits to the 5' end for translation initiation. Pea Enation Mosaic Virus (PEMV) contains two adjacent 3'CITEs in the center of its 703-nucleotide 3' untranslated region (3'UTR), the ribosome-binding, kissing-loop T-shaped structure (kl-TSS) and eukaryotic translation initiation factor 4E-binding Panicum mosaic virus-like translation enhance (PTE). We now report that PEMV contains a third, independent 3'CITE located near the 3' terminus. This 3'CITE is composed of three hairpins and two pseudoknots, similar to the TSS 3'CITE of the carmovirus Turnip crinkle virus (TCV). As with the TCV TSS, the PEMV 3'TSS is predicted to fold into a T-shaped structure that binds to 80S ribosomes and 60S ribosomal subunits. A small hairpin (kl-H) upstream of the 3'TSS contains an apical loop capable of forming a kissing-loop interaction with a 5' proximal hairpin and is critical for the accumulation of full-length PEMV in protoplasts. Although the kl-H and 3'TSS are dispensable for the translation of a reporter construct containing the complete PEMV 3'UTR in vitro, deleting the normally required kl-TSS and PTE 3'CITEs and placing the kl-H and 3'TSS proximal to the reporter termination codon restores translation to near wild-type levels. This suggests that PEMV requires three 3'CITEs for proper translation and that additional translation enhancers may have been missed if reporter constructs were used in 3'CITE identification. Importance: The rapid life cycle of viruses requires efficient translation of viral-encoded proteins. Many plant RNA viruses contain 3' cap-independent translation enhancers (3'CITEs) to effectively compete with ongoing host translation. Since only single 3'CITEs have been identified for the vast majority of individual viruses, it is widely accepted that this is sufficient for a virus's translational needs. Pea Enation Mosaic Virus possesses a ribosome-binding 3'CITE that can connect to the 5' end through an RNA-RNA interaction and an adjacent eukaryotic translation initiation factor 4E-binding 3'CITE. We report the identification of a third 3'CITE that binds weakly to ribosomes and requires an upstream hairpin to form a bridge between the 3' and 5' ends. Although both ribosome-binding 3'CITEs are critical for virus accumulation in vivo, only the CITE closest to the termination codon of a reporter open reading frame is active, suggesting that artificial constructs used for 3'CITE identification may underestimate the number of CITEs that participate in translation.

Dataset Information

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

Publications

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

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