| Southern rice black-streaked dwarf virus(SRBSDV) is a new member within the second group of the genus Fijivirus, family Reoviridae. The new rice dwarf disease caused by southern rice black-streaked dwarf virus was outbreak in the Northern of Vietnam and many provinces in Southern China in recent years and it caused severe damage on rice production. The effective new measures for the prevention and control of SRBSDV is to develop anti-SRBSDV small molecules with higher activity which are screened by anti-SRBSDV molecular compounds screening model based on protein targets. In order to reveal the genome constitution of SRBSDV, all 13 opening reading frames from of SRBSDV in the rice plants was investigated using real-time q PCR method firstly. The biological sequence information of genes with higher abundances that are S9, S8 and S10, were described, among which the viroplasm(Vps) P9-1 with the highest abundance and highly conservative property was selected as the target for further investigation. Sequence analysis of the amino acid composition concluded by bioinformatics analysis indicated that P9-1 protein had highly conserved C- and N-terminal amino acid residues and a hypervariable region that differ from 131 to 160 aa. Wild-type(WT-His-P9-1), 23 C-terminal residues truncated(TR-ΔC23-His-P9-1), and 6 N-terminal residues truncated(TR-ΔN6-His-P9-1), and Ser138 site-directed mutant(MU-138-His-P9-1) proteins were expressed and purified. WT-His-P9-1 and three different mutagenesis P9-1 proteins were subsequently selected as models for the interaction tests of P9-1 with the anti-virus small molecule dufulin(DFL) by fluorescence titration(FT) and microscale thermophoresis(MST) assays in vitro. The results indicated that the affinity of DFL with SRBSDV-P9-1 is within the micromole scale. And the mechanism of the action of DFL against SRBSDV was that DFL binds with the 23 C-terminal residues to inhibit the functioning of the Vps formation. Thus, a bioassay model that could be viable for finding new antiviral drugs was established. Finally, the binding affinity evaluation of DFL derivatives to P9-1 were studied, and some compounds with strong binding affinities to SRBSDV-P9-1 were discovered. The conclusions are shown as follows.(1) Analysis of gene expression of SRBSDV.The expression of all 13 opening reading frames of SRBSDV in rice tissues from different growth periods, field regions, rice varieties, and infection conditions were investigated. It was found that the high abundance ratios of genes expression of integrated S9-1 and S7-1, and the lowest abundance of S6 characteristics were not affected by growing period, regional, rice varieties and virus infection status. On the other hand, S8 and S2 genes also showed a slightly higher expression trend, while the other genes, such as, S1, S3, S4, S5-1, S5-2, S7-2, S9-2 and S10 showed an irregular abundance express trend.(2) Bioinformatics analysis of SRBSDV important functional genes.The sequence characteristics and classification status of genomic nucleotides and amino acids were analyzed. The physical properties and function prediction of nonstructural protein P9-1, coat protein P10, and core protein P8 were analyzed, and the full-length genome, GC content, hydrophilic or hydrophobic regions, signal peptide, secondary and tertiary structure were confirmed. Sequence homology analysis of S8, S9, and S10 were studied through Motif analysis and Blast ratio method. In order to further investigate the virus evolution trend, nucleotide mutation was analyzed by SNP, and the phylogenetic tree of S8, S9 and S10 were also established.(3) Conservative and high variable structure domain analysis of SRBSDV-P9-1.The conserved and hypervariable regions between SRBSDV-P9-1 and RBSDV-P9-1 sequences were analyzed using Gene Doc analysis. The alignment results indicated that P9-1 protein of SRBSDV and RBSDV had highly conserved C- and N-terminal amino acid residues and a hypervariable region that differ from 131 to 160 aa.(4) Expression and purification of the SRBSDV-P9-1 nonstructural proteins.Based on the bioinformatics analysis results, wild-type(WT-His-P9-1), and three mutant proteins(TR-ΔC23-His-P9-1, TR-ΔN6-His-P9-1 and MU-138-His-P9-1) were expressed and purified in the E. coli BL21(DE3) RIL.(5) Binding properties of DFL and NNM to WT-His-P9-1.The binding properties of DFL and NNM to WT-His-P9-1 were evaluated through FT and MST methods. The results indicated that the WT-His-P9-1+DFL system with a KA of 1 × 105.061 M-1(FT) and a Kd of 3.26+/-0.46 μM(MST) exhibited a higher binding affinity obviously than the WT-His-P9-1+NNM system with a KA of 1 × 104.244 M-1(FT) and a Kd of 48.40+/-6.04 μM(MST). To discover the binding sites of DFL to WT-His-P9-1, binding assays were conducted by using FT and MST method between DFL and the three different kinds of mutant proteins, namely, TR-ΔN6-His-P9-1, TR-ΔC23-His-P9-1, and MU-138-His-P9-1. The results indicated that the C-terminus 23 amino acid residues were specifically vital to the binding affinity. Thus, a new bioassay model for the screening of anti-SRBSDV bioactivities of small molecule compounds were established based on WT-His-P9-1 and TR-ΔC23-His-P9-1 proteins.(6) Screening of DFL derivatives based on SRBSDV-P9-1 target model.Based on the established bioassay model, the binding affinities of DFL derivatives to WT-His-P9-1 and TR-ΔC23-His-P9-1 proteins were investigated. Among the tested DFL derivatives, six compounds were found possessing high binding affinities(?M level) with P9-1 proteins, and their structures all contain thiophene group, together with unsubstituted phenyl moiety or phenyl ring substituted with F, Cl, NO2, CF3 moieties, etc. Besides, the binding affinities between TR-ΔC23-His-P9-1 and DFL derivatives indicated that C-terminus 23 amino acid residues were also vital to the binding affinity between DFL derivatives and WT-His-P9-1.
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