Screening Of The Key Enzyme Activity Sites Of Tembusu Virus Methyltransferase

Duck Tembusu virus?DTMUV?belongs to flavivirus genus,which encodes three structural proteins?nucleic capsid protein C,membrane protein precursor PrM,capsule protein E?and seven non-structural proteins?NS1,NS2A,NS2B,NS3,NS4A,NS4B,NS5?.Flavivirus nonstructural protein NS5 has both N-7 and 2'-O methyltransferase activities,which can regulate virus replication.Also 2'-O methylation enables viral RNA to form the same cap structure as host cell mRNA,thus escaping recognition and clearance by the host's natural immune system.At present,the research on DTMUV NS5 methyl transferase activity has not been reported.The purpose of this study was to lay a foundation for the exploration of methyltransferase deficient DTMUV as a candidate live attenuated vaccine strain by screening the key active sites of DTMUV NS5methyltransferase.1.Construction and expression of DTMUV methyltransferase recombinant plasmid and mutant recombinant plasmidThe fragment of NS5 methyltransferase active region,which is highly conserved in flavivirus,was selected for amplification.The recombinant expression plasmid pET-28a?+?-NS5-p1 was constructed by cloning the amplified gene fragment p1 to the prokaryotic expression vector pET-28a?+?and identified by double digestion.The results showed that:The recombinant expression plasmid pET-28a?+?-NS5-p1 was constructed by enzyme digestion,and two expected fragments of 5300bp and 900bp were obtained.The sequence was verified to be correct by sequencing,indicating that the construction of DTMUV methyltransferase recombinant plasmid pET-28a?+?-NS5-p1 was successful.Using pET-28a?+?-NS5-p1 as template,four sites in the tetrad structure of the methyltransferase activity section,K61-D146-K182-E218,were selected for mutation to construct mutant recombinant plasmids pET-28a?+?-NS5-K61A,pET-28a?+?-NS5-D146A,pET-28a?+?-NS5-K182A and pET-28a?+?-NS5-E218A with pET-28a?+?as carriers.This indicated that the construction of mutant recombinant plasmid was successful.The constructed recombinant plasmids pET-28a?+?-NS5-p1 and their methyltransferase activity segments were protein-induced by the tetrad recombinant mutant plasmids?pET-28a?+?-NS5-K61A,pET-28a?+?-NS5-D146A,pET-28a?+?-NS5-K182A and pET-28a?+?-NS5-E218A?.Enzyme proteins with a size of 33 kDa expressed in inclusion bodies,such as NS5-p1,NS5-K61A,NS5-D146A,NS5-K182A and NS5-E218A,were obtained.The purified inclusion body was purified by dilution refolding method according to the kit instructions.SDS-PAGE electrophoresis results showed that relatively pure soluble protein was obtained.2.Screening of key sites for DTMUV methyltransferase activityThe use of isotope ³²p radioactive labeling for screening the key activity sites of DTMUV NS5 methyltransferase showed that,when analyzing the N-7 methyltransferase activity(G³²ppp A-RNA transformed into m7G³²pppA-RNA),the enzyme activity of NS5-K61A,NS5-D146A,NS5-K182A and NS5-E218A mutation groups decreased to 25%,0%,11.5%and 33.2%,respectively,compared with that of NS5-p1 group.Meanwhile,in the analysis of 2'-O methyltransferase activity(m7G³²pppA-RNA converted to m7G³²pppAm-RNA),it was found that the enzyme activity of NS5-K61A,NS5-D146A,NS5-K182A and NS5-E218A mutation groups all decreased to 0%compared with the non-mutated group.Conclusion:The K61,D146,K182,and E218 sites of the DTMUV methyltransferase activity segment tetrad are key sites for DTMUV 2'-O methyltransferase activity,and D146 site is also the key site for DTMUV N-7 methyltransferase activity.