+ RNA Virus Invasion And Replication Of Key Protein Structure And Function Studies

Enterovirus 71(EV71)is the major pathogen responsible for outbreaks of severe hand,foot and mouth disease(HFMD).EV71 belong to the Enterovirus genus of the family Picornaviridae.EV71 can cause a wide spectrum of acute central nervous system(CNS)syndromes,including aseptic meningitis,poliomyelitis-like paralysis and acute neurogenic pulmonary edema with a high mortality.The outbreaks of EV71 infection occurred in Linyi,Shandong Province in 2007 and Fuyang,Anhui Province in 2008,initiating the EV71 pandemic in mainland China that has persisted ever since.These outbreaks made HFMD a great public health concern in China,leading to its classification as a category C communicable disease in the Chinese National communicable disease surveillance system.Science 2008,more than one million HFMD cases have been reported in China each year,according to data obtained from the Chinese National Notifiable Disease Reporting System.The genome of EV71 is a single positive-strand RNA molecule with about 7,400 ribonucletide which can encode a 250 KD polyprotein.The polyprotein need to be cleaved to 11 final products which contains three structure proteins(capsid protein):VP0,VP1 and VP3 and seven non-structure proteins:2A,2B,2C,3A,3B,3Cand 3D by protease 2A and 3C.Regular icosahedral capsid is formed by structure proteins to protect virus genome,replicase and mediate invasion.The structural protein involved in the mature of the virus,replication,innate immune escape and other critical processes of the life cycle of virus.The replication of the EV71 genome occurs in the characteristic cytoplasm membrane as other positive strand RNA virus.No-structure 2C and its precursor protein 2BC mediate the rearrangement of membrane components in the virus infected cells and the replication complex assembly based on the membrane.According to the amino acid sequence analysis,2C belongs to SF3 Helicase family of the AAA + ATPase super family.2C is also involved in the other important processes in the virus life cycle,including virus uncoating,innate immune evasion,the formation of viral capsid and so on.The membrane-bound motif at the N-terminus of 2C lead to its highly instability in vitro and make it difficult to get the crystals and its high resolution structure.Lack of a high resolution structure made it difficult to elucidate 2C activity and prevented inhibitor development.Here we report the 2.5-A-resolution crystal structure of the soluble part of EV71-2C 116-329,and its complex structure of EV71-2C 116-329 and ATP-?-S and which are the first 2C structure of the picornavirus.The structure contains an classical ATPase domain,a cysteine-rich zinc finger with an unusual fold and a C-terminal helical domain.Unlike other AAA+ ATPases,EV71 2C undergoes a C-terminus mediated self-oligomerization,which is dependent on a specific interaction between the C-terminal helix of one monomer and a deep pocket formed between the ATPase and the zinc finger domains of the neighboring monomer.Molecular sieve chromatography experiment and X-ray small Angle scattering experiment show that the oligomer of 2C is stable in aqueous solution and prove that the interaction between 2C is not caused by crystal packing.The ATPase activity of the mutations and the packing activity of the mutant recombinant virus which participate in the interaction between 2C provide that the C-terminus mediated self-oligomerization is fundamental to 2C ATPase activity and EV71 replication.As the other AAA+ATPase,2C can form a hexameric ring,so we built a hexameric model of EV71 2C 116-329 based on the crystal structure of JCV LTAg.The hexameric model provide us with important structural biology data for deeply insight into the function of 2C.Our findings suggest a strategy for inhibition of enterovirus replication by disruption of self-oligomerization interface of 2C.Human coronavirus(CoV)HKU1 is a pathogen causing acute respiratory illnesses and so far little is known about its biology.HKU1 virus uses its S1 subunit C-terminal domain(CTD)and not the N-terminal domain like other lineage A ?-CoVs to bind to its yet unknown human receptor.Here we present the crystal structure of HKU1 CTD at 1.9A resolution.The structure consists of three subdomains:core,insertion and subdomain-1(SD-1).While the structure of the core and SD-1 subdomains of HKU1 are highly similar to those of other ?-CoVs,the insertion subdomain adopts a novel fold,which is largely invisible in the cryo-EM structure of the HKU1 S trimer.We identify six residues in the insertion subdomain that are critical for binding of neutralizing antibodies and two residues essential for receptor binding.Our study contributes to a better understanding of entry,immunity,and evolution of CoV S proteins.