Studies Of Virus-like Particles And Crimean-congo Hemorrhagic Fever Virus Gn/Gc-host Interactome

Virus-like particles(VLPs)are organized particles that self-assemble from one or more viral structural proteins.They are similar to native virions,but are noninfectious due to the lack of viral genetic materials.As VLPs have the ability to induce immune responses,rapid and safer manufacturing process,they have unique advantages in the development of vaccines against emerging infectious pathogens.The baculovirusinsect cell expression system(BVES)is one of the most commonly used eukaryotic expression systems for heterologous protein expression and has been used for functional studies,antigen and vaccine preparations.We constructed several VLPs for viruses that harm human health,and preliminarily explored thier immunogenicity as vaccine candidates.Zika virus(ZIKV)is a mosquito-borne flavivirus.It has been reported that newly emerged ZIKV infection was associated with microcephaly and serious neurological complications,such as Guillain-Barre syndrome.As currently no specific vaccines and drugs are available,ZIKV infection has posed great threat to the public health.In this study,we constructed a recombinant virus expressing pr ME protein of ZIKV based on baculovirus-insect cell expression system.ZIKV VLPs self-assembled from pr M protein and E protein were prepared from infected insect cells by sucrose density gradient ultracentrifugation.The VLPs represent a promising vaccine candidate due to their potential to induce mice to produce neutralizing antibodies,specific humoral and cellular immune responses.We also constructed recombinant virus expressing ZIKV envelope protein E.The result showed that E protein assembled into VLPs similar to the natural ZIKV virions independent of pr M,and the VLPs could be released into culture supernatants.The baculovirus-expressed E protein retained fusogenic ability to induce syncytium formation,a phenomenon that was common for flaviviruses,such as dengue virus,Japanese encephalitis virus,and tick-borne encephalitis virus.Based on this phenomenon,we established an antiviral drug screening platform targeting flavivirus E protein.Poliovirus(PV)is the pathogen of poliomyelitis,which may lead to paralysis and fatality.At present,there is no specific treatment for poliomyelitis,and only vaccine can be used as prevention.Currently,live-attenuated oral poliovirus vaccine(OPV)and inactivated poliovirus vaccine(IPV)are used to defend against PV worldwide.Since 1988,the world health organization(WHO)launched the Global Polio Eradication Initiative(GPEI),countries around the world promoted the use of OPV.WHO declared PV ? eradiated at 2015 and declared PV ? eradiated at 2019.During the final stage of polio eradication,given the biosafety issues associated with OPV and IPV production,vaccines should be developed in a PV-free environment.We used baculovirus-insect cell expression system to co-express P1 and 3CD proteins of PV.P1 could be successfully cleaved into VP0,VP1 and VP3 by 3CD protease.After sucrose density gradient ultracentrifugation combined with cesium chloride density gradient ultracentrifugation,VLPs similar to native PV were purified.Crimean Congo Hemorrhagic Fever Virus(CCHFV)is a highly pathogenic virus,can cause CCHF,a tick-borne natural infectious disease.CCHFV results severe hemorrhagic fever with fatality rate ranging from 30% to 50% and is classified as biosafety level-4 pathogen.Moreover,it is difficult to amplify CCHFV through normal cell culture in vitro,which greatly limits the research progress of CCHFV.So,the mechanisms causing this damage are poorly characterized and currently there are no approved specific therapeutics or vaccines.We constructed recombinant viruses coexpressing CCHFV membrane proteins and nucleocapsid protein,or expressing membrane proteins alone in insect cells.CCHFV VLPs were purified from infected cells by sucrose density gradient ultracentrifugation.CCHFV is surrounded by a host derived envelop with spikes formed by glycoprotein Gn and Gc.In order to form infectious virions,GPC undergoes complicated cleavage and localization in cell to form structural proteins Gn and Gc,non-structural proteins GP160,GP85,GP38 and NSm.In the whole life cycle,CCHFV membrane proteins Gn and Gc play an important role in viral entry,membrane fusion,pathogenicity and immune escape.The study of CCHFV glycoprotein Gn/Gc-related host factors and the exploration of the interaction between CCHFV and host can help to understand the virus and find the key host factors for viral infection.In order to study the interaction between CCHFV glycoprotein Gn/Gc with host,firstly,we prepared monoclonal antibodies(m Abs)against glycoprotein Gn/Gc.Ectodomain of Gn/Gc expressed in E.coli were used as immunogen to immunize mice.After limited dilution and ELISA screening,combined with Western blot analysis and cell immunofluorescence,8 strains of Gn monoclonal hybridoma cells and 10 strains of Gc monoclonal hybridoma cells were obtained.Prokaryotic expressed truncated Gn/Gc proteins were used as antigens to identify the recognition epitopes.Finally,two Gn m Abs and one Gc m Ab and their recognized epitopes were identified.In order to explore Gn/Gc-interacting host proteins,we performed affinity purification-mass spectrometry analysis of viral glycoproteins immunoprecipitated from cell lysates,and identified 45 Gn/Gc-interacting host proteins.Bioinformatics analysis was performed to functionally annotate these proteins.The interaction between glycoprotein Gn/Gc with these host factors was validated by immunofluorescence.In summary,we successfully constructed ZIKV VLPs,PV VLPs,and CCHFV VLPs using BEVS,and the good immunogenicity of ZIKV VLPs showed it was a promising ZIKV vaccine candidate.We established a screening platform for antiviral drugs targeting flavivirus E protein based on syncytial phenomenon induced by baculovirus expressed flavivirus E protein.Two strains of CCHFV Gn m Abs and one strain of Gc m Ab were identified.And the identification of CCHFV glycoprotein Gn/Gc related host proteins can help us to fully understand the relationship between Gn/Gc with host and has great guiding significance to comprehensively understand CCHFV infection,subsequent functional analysis and targeting drug design.