Structural And Functional Insights Into Zika Virus Nonstructural Protein NS1

Zika virus(ZIKV),belonging to the flavivirus genus of the Flaviviridae family,was initially isolated from a sentinel rhesus macaque in 1947 in the Zika Forest of Uganda.Phylogenetic analyses of ZIKV demonstrate 2 major lineages:African and Asian.Although ZIKV has been in circulation in tropical regions of the world for more than 60 years,ZIKV infection was rarely investigated.ZIKV caused a large epidemic in South America in 2015,and subsequently spread to more than 80 countries.Since then,the mosquito-borne ZIKV has been recognized as a global public-health threat.ZIKV is transmitted among humans principally by infected Aedes aegypti and Aedes albopictus mosquitoes.ZIKV has been implicated in neurological manifestations including microcephaly in infants,and Guillain-Barre syndrome(GBS)in adults.Moreover,ZIKV can cross the blood-testis barrier(BTB),and cause testis damage,leading to male infertility in mice.In February of 2016,the World Health Organization(WHO)declared the ZIKV outbreak a "Public Health Emergency of International Concern".ZIKV has the same genome organization as other flaviviruses,such as dengue virus(DENV),yellow fever virus(YFV),West Nile virus(WNV),and Japanese encephalitis virus(JEV).The single-stranded positive-sense RNA genome of ZIKV is translated into a long polyprotein in the cytoplasm of infected cells.The polyprotein is further cleaved and processed by either host or viral proteases into three structural proteins[precursor membrane(prM)protein,capsid(C)protein,and envelope(E)protein]and seven nonstructural proteins(NS1,NS2A,NS2B,NS3,NS4A,NS4B,and NS5).The structural proteins,as the name suggests,form the virus particles.The nonstructural proteins assist in replication and packaging of the genome as well as in subverting the host pathways in favor of the virus.This thesis is focused on the ZIKV NS1 protein.Within infected cells,NS1 exists as a membrane-associated dimer after translocation into the endoplasmic reticulum(ER)lumen,where it is essential for viral genome replication.Infected cells also secrete NS1 as a hexameric lipoprotein particle(sNS1),which is involved in immune evasion and pathogenesis by interacting with components from both the host innate and adaptive immune systems,as well as other host factors.Besides,NS1 is the major antigenic marker for flaviviral infection,and it has been suggested,in combination with other markers,as a biomarker for the early detection of flavivirus infection.The molecular mechanisms of NS1 pathogenesis are relatively well established for DENV and WNV.Therefore,obtaining the crystal structure and analyzing the commonality and specificity of the structure of ZIKV NS1 is crucial to know whether previous research datas of other flavivirus can be applied to ZIKV NS1 as well.In this study,we first report the crystal structure of the full-length ZIKV NS1.The overall structure of ZIKV NS1 protein is similar to those of the DENV2 and WNV NS1,with the same protein fold and domain arrangement.They all form a homodimer,and each monomer has three domains:a β-hairpin domain,a wing domain,and a β-ladder domain.The ZIKV NS1 dimer has two different faces.On the inner face of the dimer,the β-roll,the connector subdomain,and the second half of the intertwined loop of the wing domain create discontinuous linear hydrophobic protrusions with a markedly hydrophobic surface that is the prime candidate for membrane interaction while the outer face of the dimer is polar.In the NS 1 hexamer,three dimers assemble with the polar outer faces pointing outward and the hydrophobic inner faces pointing inward so that they can interact with lipid molecules.Crystal structure of ZIKV NS1 enabled visualization of the second half of the intertwined loop in the wing domain,which was disordered in the previously reported DENV-2 and WNVNS1 crystal structures.This long loop(containing hydrophobic residues Y122,F123,and V124)forms an extended hydrophobic protrusion together with the β-roll and the connector subdomain,which is likely to involve an association with the membrane surface.In addition,the ZIKV NS1 structure reveals unique electrostatic potentials compared with the available NS1 structures of DENV-2 and WNV.The predominant features lie in the loop surface of the β-ladder domain,the inner face of the β-roll domain and the tip region of the outer face of the wing domain.The unique surface characteristics of ZIKV NS1 may be related to ZIKV neurotropism,and can be exploited in the development of novel therapeutics and diagnostic tools for ZIKV infection.We then examined the importance of the extended hydrophobic spike in the second half of the intertwined loop in membrance association in vitro and in vivo.The spike contains three hydrophobic residues Y122,F123,and V124.Liposome float up assay indicates that binding abilities of 122-124 mutant protein ZIKV NS 1-AAA and ZIKV NS1-GGG to liposomes are lower than that of ZIKV NSl-wt.The three-point mutation viruses ZIKV NS 1-AAA and ZIKV NS1-GGG constructed by reverse genetics technology,completely fail to replicate in Vero cells.Compared to wild-type ZIKV virus,the single residue mutant virus ZIKV NS1 Y122A,ZIKV NS1 F123A,and ZIKV NS1 V124A all exhibite slower replication kinetics,and lower peak titers.In a mouse model(n=5),mice challenged with wild-type virus and NS1 V124A mutant virus all died,while only two mice challenged with Y122A mutant virus died and mice challenged with F123A mutant virus all survived by contranst.This indicates the importance of NS1 Y122 and F123 residues in viral replication.MWhen any one of these two sites is mutated,the virulence of the virus diminishes or even completely loses.These above data indicate that the "spike" region formed by the three hydrophobic residues(122-124)is essential for NS1 to participate in membrane binding and subsequent viral replication.The NS1 protein is secreted by flavivirus infected cells,and the soluble forms of the protein can be detected in the bloodstream from the first day after the onset of symptoms.So serological diagnostic kits for ZIKV infection can be developed based on this feature of NS1.In addition,NS1 can also serve as a vaccine target for the prevention of flavivirus infection.Therefore,we used ZIKV NS1 protein to immunize mice and obtained 14 monoclonal antibodies and then studied their characteristics.These studies laid the foundation for further research on the molecular mechanisms and epitope informations of these antibodies,as well as the development of a serological diagnostic kit for ZIKV infection based on ZIKV NS1.