Expression, Purification And Characteristics Analysis Of Several RNA Silencing Suppressors

RNA silencing is a conserved mechanism in a wide range of eukaryotes for blockingthe invasion of foreign nucleic acids, such as viruses and transposons, which could trigger theviral RNA degradation with a homology-dependent manner. In response to these types of hostantiviral defences, it is not unexpected that viruses have evolved counteracting mechanism tointerfere with them at different levels. Many viruses have been shown to encode silencingsuppressor proteins. Different suppressors inhibit RNA silencing in different stages. Therefore,RNA silencing mechanism and the law of host-virus interactions could been uncovered bystudying on the mode of action of suppressors, and make good use of anti-viral RNA silencing.This research is mainly to identify RNA silencing suppressor activity of several viral protein,AC4, AC2and MDV068; to express and purify AC2, NS1and NS1mutants proteins usingprokaryotic expression vectors; to analysis siRNA binding activity of the purified AC2, NS1and NS1mutants proteins; to predict three dimensional structure of HVT063, a newlyidentified RNA silencing suppressor, and study its sub-cellular localization. The main resultsare as follows:(1) Screening and identification of RNA silencing suppressors. AC4and AC2proteingene from Tomato yellow leaf curl virus Shandong Tai’an isolate (TYLCV-SDTA), MDV068protein gene from Marek’s disease virus (MDV) were cloned with the specific primersaccording to the sequence information from GenBank, then inserted into binary plantexpression vector pBI121, and transformed into Agrobacterium tumefaciens strain GV3101.Silencing suppressor activity of these proteins was identified using Agrobacterium-mediatedtransient expression systems. Results showed that only AC2protein could weakly suppressRNA silencing while both AC4and MDV068protein could not inhibit RNA silencingeffectively. AC2of TYLCV-SDTA showed a low amino acid homology with most of AC2proteins which were identified as RNA silencing suppressors, but it had a high homology withC2TRAP, up to95%. (2) Expression and purification of several RNA silencing suppressors and mutantsproteins using prokaryotic expressing system. Recombinant expressing vectors of AC2, p19(from Tomato bushy stunt virus) and NS1(from Avian influenza virus) were constructedrespectively. Previous researches proved that NS1acted as RNA silencing suppressor bybinding siRNA and its alkaline amino acids were conducive to the combination between NS1protein and siRNA. To determine the key amino acids and functional domain of NS1as RNAsilencing suppressor, three single mutants of NS1, R35A (Arg was mutated to Ala), K41A(Lys was mutated to Ala), and R46A (Arg was mutated to Ala) and two deletion mutantsΔ71-230, Δ61-230were designed. All above proteins were also constructed into prokaryoticexpression vector and induced to express and purify. AC2, NS1and five NS1mutants proteinswere purified. In order to increase protein stability and solubility, expression and purificationconditions were optimized. Mice were immunized by p19protein to prepare polyclonalantibody. The antibody titer of p19was detected as1:10000.(3) Analysis of silencing suppressor activity and siRNA binding capability of NS1andits mutants. Using Agrobacterium-mediated transient expression systems, the suppressionactivity to local RNA silencing of NS1and the mutant proteins was observed. Results showedthat the35th and46th amino acid residues mutants canceled its silencing suppressor activity,revealing the two amino acids were essential for its suppressor activity; the41th Arg mutantdid not affect the suppression function. Δ71-230could inhibit single-stranded RNA inducedsilencing, but Δ61-230lost silencing suppression activity, so1-70amino acids were probablyinvolved in the formation of functional domain. To figure out whether silencing suppressionfunction of NS1is affected by siRNA binding ability, EMSA were used to study siRNAbinding ability of NS1and its mutants. Results proved that NS1and most of mutant proteinscould bind21bp siRNA in different levels except Δ61-230, and the siRNA binding efficiencyfrom high to low was Δ71-230, NS1, R35A, K41A and R46A. The silencing suppressionability of NS1and its mutants were not fully consistent to the siRNA binding activity. R35A、K41A、R46A mutants significantly reduced siRNA binding activity, but K41A mutant did notaffect its silencing suppression function. So it is proposed that binding siRNA may be not theonly way to suppress RNA silencing. Moreover, only retaining positions1-70amino acids could enhance the siRNA binding ability, which is because that amino acid deletion probablychanges the way of protein folding, making it easy to bind siRNA. In addition, experimentresults indicated AC2had a weak siRNA binding activity.(4) Three dimensional structure prediction of HVT063and its sub-cellular localization.Using I-TASSER, a protein structure forecast software, three dimensional stereo structure ofHVT063protein was predicted, which is a new RNA silencing suppressor from Turkey herpesvirus identified by our laboratory. Results displayed that the structure of HVT063proteincould be divided into N-terminal β-sheets regions (8β-sheets) and C-terminal α-helicesregions (9α-helices) and the two regions may be all related to RNA binding. It waspreliminarily suggested that HVT063protein form homodimer to bind RNA, like p19protein,through α-helix and β-sheet of two molecules. In order to further understand the role ofHVT063, fusion expression recombinant vector of GFP protein and HVT063protein wasconstructed and infiltrated into onion and Nicotiana benthamiana epidermal cells to observeits sub-cellular localization. It turned out that HVT063protein was distributed in both nucleusand cytoplasm, which was consistent with the HVT063characteristics of shuttling betweennuclear and cytoplasm mentioned in the NCBI database and was also in conformity with thesoftware predictions.