Zika Virus NS3 Protein Function And E Protein Receptor Candidate Molecule Research

zika virus(ZIKA)has prevailed around the world in recent years,threating human's health greatly.It was found that microcephaly was related to the zika infection on the pregnants.Thus,there is an urgent need to control zika virus.ZIKA genome encodes three structural proteins and seven nonstructural proteins.NS3and NS5 protein cooperate with each other to accomplish the continuous RNA replication in the host cell.This research focused on the function of ZIKA NS3,aiming to investigate the ATPase activity and dsDNA unwinding activity of ZIKA NS3 in vitro and to define the critical site responsible for the enzyme activity.We expect to learn more about the ZIKA replication mechanism and provide new target for ZIKA inhibitor development.We first constructed ZIKA NS3 prokaryotic expression vector and transformed it into E.coli to induce the expression of NS3.After being extracted,the protein was purified by Ni agarose beads.We used ATPase kit to detect the ATP hydrolysis velocity,which can reflect the ATPase activity.In order to study the dsDNA unwinding activity of NS3,we established double-strand unwinding system in vitro on the principle of FRET(Fluorescence Resonance Energy Transfer).when the dsDNA was unwinded by NS3,the fluorescence was enhanced.With the quick-change method,ZIKANS3 G198A vector was obtained at the basis of wild-type NS3(NS3WT)vector.The expression and purification of G198A were as same as NS3 WT.After being quantified by Western Blot,NS3 WT and G198A protein was compared with each other in terms of ATPase activity and dsDNA unwinding activity.Relatively pure NS3 was obtained after being expressed in E.coli and purified by Ni-agarose beads.ZIKANS3 could hydrolyze ATP in vitro(Vmax=2.76?mol/(L min),Km =0.11 mmol/L).Besides,the protein was able to unwind dsDNA in vitro with the enhancement of fluorescence.Compared with NS3 WT,the ATPase and dsDNA unwinding activity of G198A mutant was impaired obviously.ZIKA NS3 can unwind dsDNA depending on ATP hydrolysis and the 198 Glycine is critical for its enzymatic activity.ZIKA,belonging to the flavivirus genus,has a positive single-strand RNA genome.In recent years,ZIKA,which broke out globally and has damaged human's health greatly,needs to be controlled as quick as possible.ZIKA binds to the cell surface via the envelope protein(E protein),which can recognize the cell membane receptor.The virus membrane can fuse with the cell plasma membrane after the endocytosis.As a result,the virus genome can be released into the cell plasma to accomplish the infection process.So the ZIKA receptor exploration will help to clarify the molecular mechanism of ZIKA infection and provide the efficient targets for the drug design.By combining the protein biotinylation methods with themass spectra technology,we aim to identify the membrane protein which is proximal to the E protein.The protein identified will be screened to get the possible candidates,which will promote the real receptor discovery.We first established alkaline phosphatase(AP)system to see whether E protein can bind to the cell surface directly.ZIKA ECD-AP and AP protein were expressed in 293T cell and purified from the supernatant.Then the two proteins were added to bind SNB-19 and GC1 cell which can be infected by ZIKA.Finally we add substrate to start the color reaction to judge whether the binding happen.Horseradish peroxidase(HRP)system was used to biotinylate the membrane protein proximal to the E protein.Similarly,the ECD-HRP and HRP protein were expressed in 293T cell and purified in the supernatant.After the protein binding to the cell,HRP substrate was added to start the biotinylation reaction.The biotinylated protein was detected by immunofluorescence with streptavidin-488 antibody.The cell lysis was assembled and purified by the streptavidin beads after the biotinylation.Then the purified proteins were identified by the mass spectra analysis.Through the comparation between the experimental group and the control group as well as the literature reference,we acquired the possible ZIKA receptor candidates.The SNB-19 and GC1 cell in ECD-AP experimental group turned into blue-black color in AP system.The HRP assay showed that the SNB-19 and GC1 membrane in ECD-HRP experimental group have slight fluorescence.The number of biotinylated proteins identified by the mass spectra was low in both kinds of cell.However,there were still some specific proteins in the "E protein" group,which may bind to E protein.The extracellular domain of ZIKA envelope protein can bind to the surface of SNB-19 and GC1.With the HRP system and MS technology,the molecules on the cell membrane which bind to E protein can be identified.Besides,the MS anlysis result has laid foundation for ZIKA receptor investigation.