The Identification Of The RdRP Activity Of Duck Hepatitis A Virus Type 1 3D Protein And Its' Binding Site In The Genomic 3' Untranslated Region

Duck hepatitis A virus(DHAV)is the only member of Avihepatovirus in the Picornaviridae family,and belongs to positive-sense single-stranded RNA(ssRNA(+))virus.DHAV-1 is the most popular in China.Molecular biology analysis showed that 3D protein was the key enzyme for DHAV-1 genome replication,termed RNA-dependent RNA polymerase(RdRP),but this analysis has not been experimentally confirmed.In addition,studies of other ssRNA(+)viruses suggest that RdRP needs to bind to the 3' untranslated region(3' UTR)of the viral genome to initiate the synthesis of negative strand,but the binding capacity of DHAV-1 3D to the 3' UTR and the corresponding binding site remains unknown.In this study,the RdRP activity of DHAV-1 3D protein and its binding site in the 3' UTR was studied,and the following results were obtained:1.Identification of the RdRP activity of DHAV-1 3D proteinIn this study,the 3D protein of DHAV-1 H strain was expressed and purified,and SDS-PAGE reslut showed that numerous,purified and soluble 3D protein was obtained.DHAV-1 H strain 3'UTR was cloned into pMD19-T vector with a T7 promoter,and the the in vitro transcribed full-length 3' UTR RNA was obtained and in accordance with the expectation.Using 3' UTR RNA as a template for RdRP reaction,ammonium molybdate spectrophotometry and quantitative reverse transcription PCR(RT-qPCR)were applied to detect the pyrophosphate and RNA produced during the polymerization,and the result showed that the content of these two prudcution increased significantly over a period of time,indicating that the prokaryotically expressed soluble 3D protein indeed has RdRP activity.2.The study of the interaction between 3D protein and genomic 3' UTRFirst,based on the amino acid sequence of 3D protein of DHAV-1 H strain and the nucleotide sequence of 3' UTR,the interaction probability between them was predicted by RPISeq tool.It was found that DHAV-1 H strain 3D protein and 3' UTR could bind to each other.To verify this predictive result,the binding capacity between purified DHAV-1 3D protein and 3' UTR RNA was identified using the EMSA(electrophoretic mobility shift assay),and the result showed that the migration rate of the 3' UTR got slower in the presence of the 3D protein.This result suggested that the 3'UTR bound to 3D protein,which was consistent with the predicted results..3.Identification of the binding site of 3D protein in the genomic3'UTRThe 3' UTR sequence was first divided into 6 overlapping fragments termed F1,F2,F3,F4,F5,F6 respectively,and the binding capacity between 3D protein and these fragments were predicted using RPISeq.The results showed that the 3D protein could bind to the other five segments except F5.The 3' UTR fragments were first cloned into the pMD19-T vector,and the in vitro transcribed RNAs of the fragments were obtained and in accordance with the expectation.EMSA results showed that the segmentation F3,F4 and F6 of 3'UTR migrated slower in the presence of 3D protein,suggesting that the binding sites of the 3D protein in the 3' UTR are at F3,F4 and F6,ie the bases at positions 149-201 and 310-335,which is almost consistent with the predicted results.4.The disigning of small interfering RNA(siRNA)targeting the binding sites and the effect on viral replicationTwo pairs of siRN As were designed for the 149-201 bases of the binding sites,targeting the loop and stem regions respectively,and a pair of non-homologous RNAs with no homology to the DHAV=1 genome sequence were designed.Cells were infected with DHAV-1 after transfecting the siRNAs.The virus copy number was quantitatively detected by fluorescence quantitative RT-PCR targeting the viral VP1 gene,and the expression level of viral VP3 protein was detected by Western-blot with ?-actin as the internal reference.The results showed that the two pairs of siRNAs caused different degree of inhibition to viral replication and translation,and the siRNA targating the loop region resulted in significant inhibition.Therefore,the binding sites of 3D protein in the 3' UTR,especially the loop gegion,were significant for viral proliferating.In conclusion,DHAV-1 3D protein has RdRP.In addition,the binding sites of 3D protein on genomic 3' UTR are located at the 149-201 and 310-335 bases of the 3' UTR.And the binding sites play important roles in viral replication and translation.These findings are significant for elucidating the roles of 3D protein and 3' UTR in the replication mechanism of DHAV-1 genomic RNA.