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

Abstract

Background: Circularization of RNA mediated by association of translation factors and RNA elements in 5' and 3' UTRs is a common feature for translation control in eukaryotes. There is no information about translation in plant rhabdoviruses and little information is known in animal rhabdoviruses.

Methods: The role of 5' and 3' UTRs in two genes of EMDV in translation were studied using luciferase constructs and RNA structures of these sequences were analyzed by SHAPE and Inline probing.

Results: We have found that efficient translation of N and X mRNAs of nucleorhabdovirus Eggplant mottled dwarf virus (EMDV) requires elements present in both 5' and 3' UTRs. Luciferase reporter constructs containing precise 5' and 3' UTRs of the N and X genes had substantially higher translational activity compared with constructs containing only the 5' or 3' UTR. The 3'UTR of carmovirus Turnip crinkle virus, which contains a well-characterized cap-independent translation enhancer, was unable to complement the lack of EMDV 3' UTR. Addition of cap analog to luciferase constructs containing the UTRs of the N gene did not restore translation, and translation of the reporter construct in the absence of the 5' cap was higher than the capped construct. No RNA-RNA interactions between 5' and 3' UTRs were detected by EMSA or in-line cleavage structural assays. Deletion of 11 nucleotides from the 3' terminus negated the synergistic activity of the 3'UTR.

Conclusions: The results with RNA-RNA interaction suggesting that translational synergy between the UTRs may utilize alternative means. Mutation analysis in 3'UTR suggesting that the polyadenylation signal sequence contained in this location may play a critical role in translation.

Keywords: Eggplant mottled dwarf virus; Rhabdovirus; Translation.

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