Mechanistic Insights Into The Entry And Assembly Of Three Emerging Or Re-emerging Viruses

In the past two decades,outbreaks of emerging or re-emerging viral infectious diseases frequently occurred all around the globe,such as Ebola virus?EBOV?,Yellow fever virus?YFV?,African swine fever virus?ASFV?and 2019 novel coronavirus?2019-nCoV?,etc.,resulting in enormous casualties and economic losses.In order to survive,viruses must invade specific host cells to support their propagation and transmission.This process involves multiple steps,including viral attachment,entry,replication,assembly and release.Entry into host cells is the starting point of virus life cycle,and the correct viral assembly is crucial for the production of infectious virions.Thus,investigating the processes of virus entry and assembly is a prerequisite for understanding the mechanisms of virus infection,transmission and pathogenesis,which would provide important theoretical bases for prevention and control of related diseases and epidemics.In this dissertation,I will introduce the mechanistic studies on the entry and assembly processes of three representative emerging or re-emerging viruses based on cryogenic electron microscopy?Cryo-EM?approach.Firstly,we focused on the ASFV which has caused huge economic losses to China's pork industry.As the sole member of the Asfarviridae family,ASFV is a large double-stranded DNA virus?-250 nm in diameter?,consisting of multilayered structures.The huge and complicated viral particle encompasses enormous structural flexibility,rendering a formidable challenge for acquiring high-resolution structures.To tackle this problem,we utilized the block-based reconstruction algorithm newly developed for structural determination of large virus particles and successfully obtained a 4.6-A resolution structure of ASFV capsid.Based on structural analysis and comprehensive bioinformatics studies,we identified several key protein components within the capsid,including 8,280 copies of the major capsid proteins?MCP?p72,60 copies of the penton proteins and at least 8,340 copies of minor capsid proteins which contains 3 different types.The p72 protein is the major capsid component and assembles into trimeric spikes in the outer capsid shell;the penton protein forms the vertex of the icosahedral capsid;three types of minor capsid proteins form a network beneath the outer capsid shell to secure the capsid assembly by "gluing" neighboring capsomers together.These data greatly facilitate our understanding on the composition and assembly mechanism of ASFV particle and laid an important foundation for further research on the biological properties of this virus and developing antiviral countermeasures.Secondly,we explored the mechanism of Enterovirus?EV?entry into host cells.We chose Enterovirus B?EV-B?as the subject for this study which is a major group in the genus Enterovirus of Picornaviridae family,and causes severe human infectious diseases like encephalitis.The CD55 protein has been identified as an attachment receptor for EV-B.However,the precise mechanism of receptor-mediated virus entry,especially the triggering for uncoating process,remain obscure.Initially,we identified human neonatal Fc receptor?FcRn?as a universal receptor for a large group of EV-B viruses,by CRISPR-Cas9 library-based screening.Then,with the highly virulent Echovirus 6?Echo 6?as a model,we reconstituted its uncoating process in vitro triggered by FcRn-decorated liposomes.To understand the structural basis of interactions between virus and its receptors,we determined a series of high-resolution structures of Echo 6 virus particle and its complex with either CD55 or FcRn receptors at both neutral and acidic conditions.These structures revealed that FcRn binds to the virus particle in the special "canyon" through its FCGRT subunit,while CD5 5 binds across the particle outside of the "canyon ".As an attachment receptor,CD55 does not induce obvious conformational changes of the virus,whereas the binding of FcRn triggers efficient release of the " pocket factor”under acidic conditions to initiate the uncoating process of viral particle.This study established a unified model for two-receptor mediated entry of EV-B,which would provide important basis for developing antiviral drugs by intercepting virus entry.Finally,we also made great efforts to study the virion structure of YFV which is the causative agent of the severe YF epidemics in Brazil and other American countries.YFV is the founding member of the Flavivirus genus in the family of Flaviviridae,which can cause severe diseases such as fever with jaundice and hemorrhage.The YFV particle contains only one major envelope protein?E?embedded in its membrane,which is responsible for virus attachment and entry.The receptor of YFV E protein is not known yet.In this project,we determined the structure of yellow fever live-attenuated vaccine?YF₁7D?mature viral particle at near-atomic resolution by Cryo-EM single particle reconstruction.This structure revealed unique interfaces for YFV E protein assembly compared with other flaviviruses,which contribute to the stability of YFV virions.Besides,we identified several key sites that may potentially be engineered to enhance the stability of YF 17D.In addition,the 150-loop of YFV E protein lacks the canonical glycosylation modifications as observed in other flaviviruses.These glycans have been shown to be involved in receptor recognition,which might thus affect the host tropism and virulence of YFV.These findings provide novel structural insights into the stability and virulence of YFV,which would be of great significance for the development of vaccines,therapeutics and YFV-specific detection systems.In summary,we investigated the entry and assembly mechanisms of three emerging or re-emerging viruses by structural,biochemical and cell-based approaches.These works would provide a solid basis for developing novel antiviral drugs and therapeutic measures.