Screening Of Flavivirus NS2B3 Protease Inhibitors And Study Of Their Molecular Mechanism

Flaviviruses,including Zika virus(ZIKV),Dengue virus(DENV),West Nile virus(WNV)and Japanese Encephalitis virus(JEV),are enveloped,positive-sense singlestranded RNA viruses.Flaviviruses are often transmitted to human beings by infected mosquitoes or ticks,which have always been a great threat to human health.For example,the outbreak of Zika virus in 2015 in American,has infected thousands of people,and has caused serious diseases like Guillain-Barré syndrome(GBS),a severe nervous system disease.Zika virus may also cause the microcephaly in infants.Each year,over 100 million people are infected by Dengue virus and thousands of them die of severe dengue hemorrhagic fever(DHF).However,there are no effective vaccines or drugs for these viruses.Although there is one vaccine for DENY approved by WHO,its application is limited.Therefore,it is very important to develop new vaccines and screen new drugs against flavivirus.Flavivirus genome is approximately 11 kb long,encoding 3 structural proteins and 7 non-structural proteins.The non-structural protein 3(NS3)is a multi-functional protein,which consists of N-terminal protease domain and C-terminal helicase domain.It has been proved that a cofactor region,containing～50 amino acids,in non-structural protein 2B(NS2B),is essential for the protease activity of NS3,so the protease is also called NS2B3 protease.NS2B3 protease is an important target for screening antiflavivirus drugs.However,despite many efforts to identify new molecule inhibitors that target NS2B3 protease,there have been no inhibitors approved into clinical application.In this study,we designed a new screening assay based on the self-cleavage activity of NS2AB3 to identify small molecules that inhibit the protease activity of NS2B3 protease.From an inhibitor library containing 78 inhibitors,we identified 5 potent proteasomal inhibitors that effectively inhibited the self-cleavage of NS2AB3 at nano-molar range.These 5 inhibitors also inhibited Zika and Dengue virus replication,with EC50 from 8 nM to 140 nM,independently.Bortezomib was the most potent inhibitor with an EC50 at 8 nM.The inhibition of virus genome RNA replication by these inhibitors was also verified by using a Zika virus replicon-luciferase assay and ds-RNA staining assay.Next,by using in vitro protease assay and thermo shift assay,we found that these 5 inhibitors could inhibit and interact with the NS2B3 protease,but at very high concentrations.The IC50 values from the protease assay reached to milli-molar level,which were almost 1000 folds compared to the EC50 values calculated from the virus inhibition assay in the cell.These results suggested that other host factors or cell pathways may participated in the inhibition of NS2AB3 self-cleavage.Interestingly,using immune-fluorescence,sucrose gradient centrifugation and immune-precipitation assay,we found that the NS2B3 was hyper-ubiquitinated and aggregated into aggresomes in the cell after Bortezomib treatment,leading to the attenuation of its protease activity.These results indicated that Bortezomib inhibited NS2B3 protease activity by targeting the ubiquitin-proteasome pathway.Protein ubiquitylation is mediated by the E3 ligases,and the E3 ligase of ZIKV NS2B3 has never been identified.We next identified two E3 ligases,i.e.,HRD1 and RNF126,which can interact with and ubiquitinate ZIKV/DENV NS2B3.HRD1 and RNF126 are the two E3 ligases,which play a necessary role in the ER-associated degradation(ERAD)pathway.We found that over-expression of HRD1 or RNF126 inhibited the self-cleavage of NS2AB3 and viral replication,suggesting that ERAD was important in the inhibition of flavivirus replication by Bortezomib.In summary,our work provided a new screening method to identify potential drugs targeting the NS2B3 protease,and explained a novel underlying molecular mechanism of the inhibition of NS2B3 protease by Bortezomib and providing new insights for the drug screening and development of anti-flavivirus therapeutics.