Study Of The Relationship Between RNA Silencing Suppressor Activity And Small RNA Binding Ability Of Cucumber Mosaic Virus2b Protein

Upon virus infection，small interfering RNA (siRNA)-mediated host anti-viraldefense（named as host anti-viral RNA silencing） would be triggered to attenuate orsuppress virus infection. To overcome anti-viral RNA silencing, plant viruses usuallyencode one or more RNA silencing suppressors which inhibit functions of RNAsilencing, which would benefit virus infection and replication. Cucumber mosaic virus(CMV) encoded2b protein was one of the earliest identified viral suppressors of RNAsilencing (VSR). Interestingly, the VSR abilities of the2b proteins encoded by CMVsubgroup I and subgroup II are differential. Furthermore,2b encoded by CMVsubgroup II can’t disturb microRNA pathway. But the mechanism underlying theforementioned difference remains to be elucidated.It is generally considered that competitive binding with small RNAs （sRNA）isthe main manner, by which CMV2b utilized to inhibit RNA silencing. In this study, weused2b proteins encoded by subgroup I CMV Fny-CMV and subgroup II CMVLS-CMV as research objects to uncover the mechanism leading to the difference inVSR ability between2b proteins encoded by CMV subgroups I and II. To discover therelationship between VSR ability and sRNA binding ability，we cloned Fny2b andLS2b into pGEX4T-1to express them in Escherichia coli, then we purified theexpressed Fny2b and LS2b and make them bind sRNA in vitro. The result ofelectrophoresis mobility shift assay(EMSA) showed that Fny2b had a much higheraffinity to double-stranded miRNA(ds-miRNA), double-stranded siRNA(ds-siRNA)and single-stranded miRNA(ss-miRNA) than LS2b. Based on this finding, wesuggested that the difference in VSR activity between Fny2b and LS2b was connectedto their differential sRNA-binding abilities.In this study, we also studied the vital domains located in Fny2b and LS2b forsRNA binding, we constituted recombinant2b proteins via exchanging Fny2b andLS2b at multiple sites. After sRNA-2b binding in vitro and subsequent EMSAdetection, we found that exchange of N-terminal12amino acids didn’t affect sRNA binding ability of Fny2b and LS2b; whereas when Fny2b N-terminal47amino acidswas replaced with LS2b N-terminal47amino acids, the recombinant2b proteinshowed an equally weak sRNA binding ability to LS2b, which allows us to proposethat the difference in amino acid residues between the N-terminal13-47of Fny2band LS2b would be contributed to the differential VSR activities between Fny2b andLS2b. Additionally, we substituted arginine at positions33,36, or46with alanine, andproline at position41with alanine as well, singly in Fny2b and LS2b, and tested VSRactivities of these mutants. All these mutants impaired VSR activities of Fny2b andLS2b at a varied extent. Amongst, both Arg33and Arg36play an important role insuppression of host RNA silencing, especially Arg36.In conclusion, this work found that the difference of VSR ability between F ny2band LS2b was connected to their differential sRNA binding ability, which is associatedwith the the N-terminal13to47sequences of Fny2b and LS2b.