Efficient translation of Eggplant mottled dwarf nucleorhabdovirus N and X genes requires both 5′ and 3′ UTRs

Fig. 1. A Schematic representation genomic organization of EMDV containing 7 ORF (+ strand of virus genome). B Schematic representation of Luciferase constructs for N gene of EMDV. N gene constructs contain 80 and 180 nt of 5′ N gene in 5′ side and 3′UTR of N gene in 3′ side of the luciferase gene, and luciferase construct contains precise 80 nt of 5′ N gene in 5′ side and 3′ UTR of TCV in 3′ side of the luciferase gene. Schematic representation of luciferase construct contains N UTRs and Predicted structure for 3′ UTR of N gene which selected region of 11 nt in mutagenesis has been shown. C Relative In vitro translation activity in luciferase constructs. Luciferase activity in luciferase reporter transcript containing the 5′ and 3′ fragments from N gene include construct contain TCV (393) 3′UTR capped construct and mutants (mutant of the N5′80-fluc-N3′UTR construct with 11 nt (position 1804–1814) deletion from the end of 3′ UTR). D In vitro translation of EMDV N gene with different amount of RNA in wheat germ extract. Lane 1, control without any RNA Lane 2, PEMV2′s RNA as control for translation system. Lane 3 and lane 4, 0.5 μl and, 2 μl of N gene RNA

Fig. 2. A Schematic representation Luciferase constructs for X gene of EMDV. X gene constructs contain 8 and 140 nt from 5′ X gene and 3′ UTR of X gene in 5′ and 3′ sides of the luciferase gene. B Luciferase activity in luciferase reporter transcript containing the 5′ and 3′ fragments from X gene. C In vitro translation of EMDV X gene in wheat germ extract with different amount of RNA. Lane 1, 2 μl and lane 2, 0.5 μl of RNA. Lane 3, control without any RNA Lane 4, PEMV’s RNA as control

Fig. 3. A RNA structure prediction by in-line probing for 5′ and 3′ UTRs of EMDV-N gene and schematic of putative structure of 5′ and 3′ UTR RNA by in-line probing. B SHAPE analysis of N gene UTRs and Putative structure of 5′ and 3′ UTR of EMDV-N gene using SHAPE. High reactivity/cleavage showed by red and lower reactivity/cleavage showed by green color