Structural And Functional Studies On Non-structural 2C Protein Of Hepatitis A Virus

Hepatitis A virus(HAV)is the sole member of the Hepatovirus genus in Picornaviridae,underscoring its uniqueness in this family.HAV remains a major pathogen causing communicable liver disease worldwide.Although HAV infection is generally mild,a small portion of HAV patients(particular aged over 50)could die from fulminant hepatitis.Available HAV vaccines can provide efficient and lasting immune protection.Nevertheless,occasional HAV outbreaks still occur nowadays even in developed countries with good sanitary and hygienic conditions.To rapidly contain future outbreaks,treating fulminant hepatitis A patients as well as controlling potential vaccine-escape variants,development of effective anti-HAV drugs remain a great value in disease control.Distinct from many picornaviruses,the structure of HAV particles shares features of both standard picornavirus and insect picorna-like viruses.The icosahedral capsid of HAV(?27 nm in diameter)is remarkably smooth and without canyons,which are consistent to its ability to withstand high temperature and low pH environments.The genome of HAV is a?7.5 kb plus-sense single-stranded RNA comprising a 5'untranslated region(UTR),a large open reading frame(ORF),a 3'-UTR followed by a poly(A)tail.The ORF encodes a large polyprotein(-250 kDa)that is processed co-and post-translationally by viral and cellular proteases,yielding mature structural proteins(VP1-VP4),nonstructural proteins(2A,2B,2C,3A,3B,3C and 3D)as well as multiple functional precursors.Distinct from typical picornaviruses,the 2A protein of HAV does not functions as a protease;instead,it is a part of VP1.The 3C and 3ABC proteins of HAV are proteases responsible for most processing steps.As a SF3 helicase family member,it is not surprising that HAV 2C has ATPase and RNA binding activities,but the duplex unwinding activity of HAV 2C has not been reported,in contrast to enterovirus 71 2C.Both full-length HAV 2C and a truncation lacking the N-terminal membrane binding motif exhibited RNA binding activity,implying that the RNA binding site does not reside in the N-terminal region.HAV 2C recognizes the 3'-terminal untranslated region of negative-strand viral RNA,suggesting it might anchor the negative-strand RNA,the template for positive-strand RNA synthesis,to intracellular membranes.In previous studies,we determined crystal structures of enterovirus 71 2C and PV 2C lacking the N-terminal amphipathic helix.We demonstrated that enterovirus 2Cs are composed an ATPase domain followed by a zinc-finger and a C-terminal amphipathic helix.The extreme C-terminal helix of enterovirus 2C occupies a hydrophobic pocket of the adjacent 2C and mediates a specific 2C-2C interaction essential to the ATPase activity of 2C and virus replication.We proposed a hexameric-ring model for enterovirus 2Cs,which contains the negatively charged central pore.Whether those features of enterovirus 2C preserves in the ancestral HAV 2C is important to understand the molecular evolution of picornaviral 2C proteins.Here,we characterized HAV 2C protein structurally,biochemically and functionally.We determined the crystal structure of a soluble HAV 2C containing the entire ATPase domain,a region equivalent to enterovirus 2C zinc-finger(ZFER)and a C-terminal amphipathic helix.The C-terminal helix of HAV 2C occupies a hydrophobic pocket(Pocket)of the adjacent 2C which mediates a specific 2C-2C interaction.Mutagenesis studies demonstrated the PBD/Pocket interaction is essential to self-oligomerization and ATPase activity of 2C as well as HAV replication.Strikingly,in our biochemical characterization,we found HAV 2C exhibits ribonuclease activity on single-stranded RNA with a preference for poly-U segments,and the ribonuclease activity is independent of the ATPase activity of HAV 2C.We futher identified three acidic residues essential to the ribonuclease activity and showed those residues are fundamental to HAV replication.Given these acidic residues reside in a conserved region,we tested ribonuclease activity of a variety picornavirus 2Cs,including EV71 2C,FMDV 2C,CVB3 2C,PV 2C and HRV 2C,and confirmed they all share the similar activity.This study determined the c-terminal crystal structure of the 2C protein of hepatitis A virus and discovered for the first time that the 2C protein has single-stranded nuclease activity.We found the key amino acids responsible for ribonucleic acid catalysis.The research on the function has laid a good foundation and provides a good structure and function foundation for the design of drugs targeting 2C protein.The pandemic caused by the SARS-CoV-2 is continuing to erupt and spread as never before.So far,there is still no effective treatment or prevention means to slow down or stop this crisis.SARS-CoV-2 papain-like protein(PLpro)plays a vital role in virus replication and immune evasion,and is a very effective target for drug-design.Given that the SARS-CoV-2 and SARS-CoV PLpro proteases have significant homology,the development of inhibitors for SARS-CoV PLpro is a promising starting point for therapeutic development.In this study,we targeted PLpro to find inhibitors that can target the protein,so as to provide ideas for drug design targeting the protein.First,we determined the monomer structure of SARS-CoV-2 PLpro mutant C111S,which has many structural features identical to SARS-CoV PLpro.The crystal shape of this protease has the characteristics of unique accumulation and high solvent content.In the end,we obtained a crystal structure with a resolution of 2.5(?).Therefore,this provides a good possibility for fragment-based screening using crystallographic methods.We then characterized the protease activity of PLpro in cleaving synthetic peptides containing nsp2/nsp3 linkers.Finally,we proved that an effective SARS-CoV PLpro inhibitor GRL0617 is also very effective in inhibiting the protease activity of SARS-CoV-2,with an IC50 of 2.2±0.3?M.Then we analyzed the structure of the GRL0617 and SARS-CoV2 PLpro complex with a resolution of 2.6 A.The structure showed that the inhibitor occupies the S3-S4 pocket of the protein substrate binding region.The binding of GRL0617 can induce the closure of the BL2 loop and narrow the substrate binding pocket,while the binding of the tetrapeptide substrate expands the crack.Therefore,our results clarify the molecular mechanism of GRL0617 inhibition,that is,GRL0617 not only occupies the substrate pocket,but also seals the entrance of the substrate binding crack,thereby preventing the binding of the substrate's LXGG motif,and ultimately leading to the loss of protease function.