Identification of an Internal RNA Element Essential for Replication and Translational Enhancement of Tobacco Necrosis Virus AC

Heng Pu,Jialin Yu,Chenggui Han,Dawei Li,Jiang Li,Zhengguang Zhang

doi:10.1371/journal.pone.0057938

Heng Pu, Jialin Yu + Show 4 more

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https://doi.org/10.1371/journal.pone.0057938

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Journal: PLoS ONE	Publication Date: Feb 27, 2013
Citations: 6	License type: CC BY 4.0

Affiliation: China Agricultural University

Abstract

Different regulatory elements function are involved in plant virus gene expression and replication by long-distance RNA-RNA interactions. A cap-independent functional element of the Barley yellow dwarf virus (BYDV) – like translational enhancer (BTE) is present in Tobacco necrosis virus A (TNV-A), a Necrovirus member in the Tombusviridae family. In this paper, an RNA stretch flanking the 5′ proximal end of the TNV-AC coat protein (CP) gene was shown to be essential for viral replication in Chenopodium amaranticolor plants and tobacco cells. This internal sequence functioned in transient expression of β-glucuronidase (GUS) when present at either the 5′ or 3′ sides of the GUS open reading frame. Serial deletion analyses revealed that nine nucleotides from nt 2609 to 2617 (−3 to +6 of the CP initiation site) within TNV-AC RNA are indispensable for viral replication in whole plants and tobacco cells. Fusion of this RNA element in mRNAs translated in tobacco cells resulted in a remarkable enhancement of luciferase expression from in vitro synthesised chimaeric RNAs or DNA expression vectors. Interestingly, the element also exhibited increased translational activity when fused downstream of the reporter genes, although the efficiency was lower than with upstream fusions. These results provide evidence that an internal RNA element in the genomic (g) RNA of TNV-AC, ranging approximately from nt 2543 to 2617, plays a bifunctional role in viral replication and translation enhancement during infection, and that this element may use novel strategies differing from those previously reported for other viruses.

Highlights

Positive-stranded RNA viruses often harbour RNA elements within their genomic (g) RNAs that mediate a variety of fundamental viral processes
At 4 dpi, the results revealed that deletions in the 59 end of the coding region eliminated symptoms and greatly reduced RNA synthesis, compared to the internal or the 39 end deletions TNVAC gRNA, which had elevated RNA levels similar to those of wild type TNV-AC (Fig. 1B and C, S1B and S1C)
These results suggest the existence of a gRNA element near the 59proximal end of the coat protein (CP) gene that is responsible for viral RNA synthesis

Summary

Introduction

Positive-stranded RNA viruses often harbour RNA elements within their genomic (g) RNAs that mediate a variety of fundamental viral processes. Long-range interactions between internal gRNA segments are required for initiation of subgenomic (sg) mRNA transcription in various plant viruses, including Red clover necrotic mosaic virus (Dianthovirus) [5], Cucumber leaf spot virus (Aureusvirus) [6], Potato virus X (Potexvirus) [7] and TBSV [8,9,10]. These results demonstrate the prevalence and fundamental importance of regulatory RNA elements for diverse reproductive strategies. These interactions have been well studied to understand the mechanistic features of virus reproduction [15,16,17], in which the sgRNA2 leader of TNV-A can interact synergistically with the BYDV 39 element to promote in vitro translation [17]

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Conclusion