Study On The Influence Of The Extreme Sequences On Protein Expression And Regulation Of Rabbit Hemorrhagic Disease Virus

【Objective】At present, we know less about the genome structure, function andpathogenic mechanism of Rabbit hemorrhagic disease virus(RHDV),which are the basis ofeffective control RHD. Therefore, this article is trying to explore the expression andregulation mechanism of RHDV genome through analysis the function of the extremesequence of RHDV genome, and hope to provide some useful clues for further exploring thetranslation mechanism of RHDV.【Method and Result】In order to evaluate the translation initiation function of the5'endsequence of RHDV genome, a bicistronic reporter plasmid was constructed by the insertion ofa cDNA corresponding to the RHDV5' extreme sequence (nts1to429) between two reportergene sequences, the first encoding CAT protein and the second encoding LUC protein. Thebicistronic plasmids were individually added to TNT rabbit reticulocyte lysate (RRL) system(Promega),and then the reporter proteins translated in vitro were evaluated by measuringLUC activity. Internal initiation activity of the different sequences (5'/3') included betweentwo reporters genes was estimated by the accumulation of LUC. Our results showed that allplasmids containing the RHDV5' extreme sequence in sense orientation allowed theexpression of LUC, indicating that the5' extreme sequence of RHDV could direct translationinitiation in vivo.To confirm and extend the results from these assays in vitro, diffent types ofmonocistrons were constructed and tested by transient-expression experiments in mammaliancells. Our results showed that the5'extreme sequence of RHDV could produce efficient LUCexpression in host cells. Though the expression level of5' end sequence of RHDV was lowerthan that of EMCV IRES clearly, these results still indicated that RHDV5'end sequence had translation initiation function in vivo.To further characterize the regulation of3' NCR on the function of RHDV genome,additional bicistronic reporter plasmids were constructed, containing RHDV3'NCR. Thebicistronic plasmids were individually added to TNT rabbit reticulocyte lysate (RRL) system(Promega),and then the reporter proteins translated in vitro were evaluated by measuringLUC activity. Internal initiation activity of the different sequences included between the tworeporter genes was estimated by the expression of LUC. Moreover, the bicistronic plasmidswere transfected into vFT7infected BHK-21cells, in order to evaluate internal initiationactivity in vivo, and the expression of LUC protein expressed from the different constructswas determined through detecting LUC activity. Our results indicated that the presence of3'NCR was not required for translation initiation,but it indeed positively modulated thefunction of the5' end sequence of RHDV.To evaluate the influence of the5'/3' extreme sequence on the expression of RHDVthrough gene level, the RHDV replicon carrying LUC gene was constructed, and then seriesreplicon mutants were also constructed. RK13cells were transfected with these repliconmutants, and the affection of deleted mutation on the expression of RHDV genome wasanalyzed through measuring LUC activity. The experimental results showed that5'endseqnence (429bp) took a key role during the expression of RHDV genome, which isconsistent to above results.【Conclusion】RHDV5' extreme sequence indeed positively regulated the expression ofLUC,and then,it could direct translation initiation in vivo.3'NCR was not required fortranslation initiation,but it indeed positively modulated the function of the5' end sequence ofRHDV.5'NCR and NSP1sequence was the key sequence,which could matain translationinitiation function of the5'end sequence in vivo.