Identification of an internal RNA element essential for replication and translational enhancement of tobacco necrosis virus A(C)

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-A(C) 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-A(C) 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-A(C), 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.