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Background: Polyadenylation influences many aspects of mRNA as well as viral RNA. variable polyadenylation at the 3' end have been reported in RNA viruses. It is interesting to identify the characteristic and potential role of 3' polyadenylation of Wheat yellow mosaic virus (WYMV), which has been reported to contain two genomic RNAs with 3' poly(A) tails and caused severe disease on wheat in East Asia region., Methods: 3' RACE was used to identify sequences of the 3' end in WYMV RNAs from naturally infected wheat by WYMV. In vitro translation assay was performed to analyze effect of UTRs of WYMV with or without 3'polyadenylation on translation. In vitro replication mediated by WYMV NIb protein were performed to evaluate effect of variable polyadenylation on replication., Results: Variable polyadenylation in WYMV RNAs was identified via 3' RACE. WYMV RNAs in naturally infected wheat in China simultaneously present with regions of long, short, or no adenylation at the 3' ends. The effects of variable polyadenylation on translation and replication of WYMV RNAs were evaluated. 5'UTR and 3'UTR of WYMV RNA1 or RNA2 synergistically enhanced the translation of the firefly luciferase (Fluc) gene in in vitro WGE system, whereas additional adenylates had an oppositive effect on this enhancement on translation mediated by UTRs of WYMV. Additional adenylates remarkably inhibited the synthesis of complementary strand from viral genome RNA during the in vitro replication mediated by WYMV NIb protein., Conclusions: 3' end of WYMV RNAs present variable polyadenylation even no polyadenylation. 3' polyadenylation have opposite effect on translation mediated by UTRs of WYMV RNA1 or RNA2. 3' polyadenylation have negative effect on minus-strand synthesis of WYMV RNA in vitro. Variable polyadenylation of WYMV RNAs may provide sufficient selection on the template for translation and replication.