Differential Regulation Mechanism On The Cap-independent Translation In Wheat Yellow Mosaic Virus

Wheat yellow mosaic virus?WYMV?,a member of the genus Bymovirus in the family Potyviridae,is 3‘polyadenylated and contains a 5‘genome-linked protein VPg.WYMV has two positive single-strand RNAs.7.6 kb of RNA1 encodes a polyprotein of 270 KDa and 3.6kb of RNA2 encodes a polyprotein of 100 KDa.A total of 10 mature functional proteins were produced by protease cleavage on two polyproteins,and the additional P3N-PIPO protein may be produced by a transcriptional frameshift strategy.This study was mainly focused on the regulation mechanism on cap-independent translation in WYMV,includingthe core cis-elements of WYMV RNA1 and RNA2,and differential regulation mechanism on the cap-independent translation in differential WYMV.Genome sequence of Wheat yellow mosaic virus?WYMV?isolated from Tai'an city and Linyi city were newly cloned.Nucleotide sequences of 5‘untranslated regions among different WYMV isolates have a relatively higher mutation rate which is suggested that the5‘UTR may play an important role in WYMV gene expression regulation.Using in vitro translation system of wheat germ extract and the firefly luciferin vector?Fluc?,the 5‘UTR of three isolates?TADWK,LYJN,HC?of RNA1 and RNA2 was analyzed for regulation mechanism on the cap-independent translation.The results showed that the 5‘UTRs of the three isolates WYMV RNA1 and RNA2 all had cap-independent translation enhancer activity.The translation regulation efficiency of 5‘UTR in RNA1 of the three isolates is basically the same,while the translation regulation efficiency of 5‘UTR in RNA2 of the three isolates has the difference of up to 7-folds.We analyzed the regulation mechanism on the cap-independent translation in WYMV-HC RNA1.In this study,IRES activity of the 5‘UTR in WYMV RNA1 was identified,and the IRES activity of the 5‘UTR could be enhanced to 3.15-fold in the presence of the 3‘UTR.The core elements of IRES within the 5‘UTR of RNA1 are mainly located from position 20 to position 130 by gradient deletion experiment.In-line probing was used to analyze the structure of the 5‘UTR of RNA1.The core elements of IRES have two hairpins?H1 and H2?andlinker region?LR1?.In the presence of the 3‘UTR,preliminary long-distance RNA–RNA interaction between 5‘UTR and 3‘UTR were mapped.The 3‘UTR synergistically enhanced this IRES activity via long-distance RNA–RNA interaction between ⁸⁰CU and⁷⁵⁷⁴AG by EMSA and in vitro translation analysis.The structural stability and 3‘nucleotide specificity of the H1 upper loop,length of discontinuous stems and 5‘nucleotide specificity of the H2 upper loopcan positively regulating IRES activity.Trans-competition assay found that the IRES of WYMV RNA1 5‘UTR is in the manner of eIF4E-dependent,and the target site of the eIF4E within the 5‘UTR of WYMV RNA1 is the top loop of H2,especially the¹¹⁴CUUUCC.In addition,the In-line probing showed that the cytosines(C⁵⁵,C⁶⁶,C¹⁰⁵,and C¹⁰⁸)in the hairpins H1 and H2 and the guanines(G⁷³,G⁷⁹,and G⁸⁵)in LR1 areclosed.Through a series of mutant in vitro translation,it was found that the 4C and 3G form discontinuous base pairing to maintain the dynamic equilibrium state.Dynamic base pairs between uncleaved C⁵⁵ and C⁶⁶ in H1 and uncleaved guanines(G⁷³,G⁷⁹ andG⁸⁵)in LR1 have positive effects on the IRES activity of the RNA1 5‘UTR,and dynamic base pairs between uncleaved C¹⁰⁵and C¹⁰⁸ in H2 and uncleaved guanines(G⁷³,G⁷⁹ and G⁸⁵)in LR1 negatively regulate the IRES activity of the RNA1 5‘UTR.Dynamic base pairs among uncleaved cytosines(C⁵⁵,C⁶⁶,C¹⁰⁵,C¹⁰⁸)in H1/H2 and uncleaved guanines(G⁷³,G⁷⁹ and G⁸⁵)in LR1maintain a tertiary equilibrium state to ensure the IRES activity of the RNA1 5‘UTR at a suitable level.This is the first time that a tertiary equilibrium state is found in the IRES element,and it is also suggested that robustness not at the maximum level of translation is the selection target during evolution of WYMV RNA1.Due to the significant differences in cap-independent translation enhancer activity of RNA2 5‘UTR in different WYMV isolates,WYMV-HC and WYMV-LYJN RNA2 were used for comparative studies.In this study,IRES activity of the 5‘UTR in WYMV RNA2 was identified,and the IRES activity of the 5‘UTR could be enhanced to 1.5-fold in the presence of the 3‘UTR.The core elements of IRES within the 5‘UTR of WYMV-HC RNA2 and WYMV-LYNJ RNA2 are mainly located from position 20 to position 150 andposition 30 to position 150 by gradient deletion experiment.In-line probing analyzed the structure of WYMV-HC and WYMV-LYJN RNA2 5‘UTR,showing that there are two difference regions between the WYMV-HC and WYMV-LYJN RNA2 5‘UTR,including the structural stability of the hairpin H1 and nucleotide specificity of the hairpin H2 upper loop.A series of mutation experiments showed that these two difference regions were main factors resulting into the difference of IRES activity of RNA2 5‘UTR between the WYMV-HC and WYMV-LYJN.It was also found that there was a relatively conserved top loop in H1 of the two WYMV RNA25‘UTR,and this conserved loop was play an important role on thecap-independent translation.Trans-competition assay found the IRES of WYMV RNA2 5‘UTR is also in the manner of eIF4E-dependent,and the target site of the eIF4E within the 5‘UTR of WYMV-HC RNA2 is the top loop of H1,but the target site of the eIF4E within the 5‘UTR of WYMV-LYJN RNA2is the top loop of H1 and H2.It is suggested that the basic difference of the translation regulation efficiency in WYMV-HC and WYMV-LYJN are the difference in the number of target sites of the eIF4E,which may cause different eIF4E binding efficiencies under the same conditions.In this study,the IRES regulatory mechanism of the only biviral genus?genus Bymovirus?in the family Potyviridae was analyzed.For WYMV RNA1,a tertiary equilibrium state of the IRES in the RNA1 5‘UTR were found to regulate translation at a suitable level.For WYMV RNA2,it is mainly focused on the the differential regulation model of the RNA2 5‘UTR between different WYMV isolates.It has enriched the research depth of IRES of plant RNA virus,and provided theoretical basis and targets on control of WYMV.