The Fold-switching Mechanism Of SARS-CoV-2 ORF9b And Molecular Basis Of The Receptor Binding Specificity Of The Hemagglutinin Of Human-infecting H5N8

Respiratory viruses can invade the respiratory tract and proliferate in the epithelial cells of the respiratory mucosa and cause local infection of the respiratory tract or lesions of tissues and organs outside the respiratory tract.The respiratory diseases caused by respiratory viruses have the characteristics of strong susceptibility,fast transmission speed and being difficult to control,which seriously endanger human life and health and cause huge economic losses.This article focuses on the research of SARS-CoV-2 and influenza virus for viral infection and pathogenesis.Recent studies have shown that SARS-CoV-2 Alpha mutant inhibits natural immune responses more effectively due to increased expression of innate immune antagonists such as N,ORF9b and ORF6,which indicates that variations outside the spike region also play a key role in the SARS-CoV-2 viral evolution.In addition,anti-ORF9b antibodies have been identified in the sera of SARS-CoV and SARS-CoV-2 patients,indicating the significance of ORF9b in virus diagnosis and immune response.Thus,understanding the molecular mechanism of ORF9b is crucial,so we carried out the first part of this thesis.In the first part of this thesis,we identified the dimeric structures of SARS-CoV-2 ORF9b using two kinds of preparations,in vitro expressed soluble and refolded forms,which showed a long hydrophobic tunnel containing a lipid that is crucial for the dimeric conformation.We also determined that the lipid ligands in ORF9b expressed in different expression systems are the same(monoglycerides)using liquid chromatography with tandem mass spectrometry(LC-MS/MS)analysis.Eight phosphorylation sites of ORF9b were identified by LC-MS/MS.Point mutation and thermal stability experiments suggest that the S50 and S53 sites are important for the structural stability of ORF9b dimer,and the two sites were highly conserved,which may be a potential target for drug development.Several studies have shown that SARS-CoV-2 ORF9b monomer can bind TOM70 to inhibit the innate immune response.We proposed a model of multifunctional ORF9b with distinct structures,suggesting that ORF9b is a fold-switching protein.Specifically,the ORF9b can interact with Tom70 with partially long a helix conformation to suppress the innate immune response,whereas the ORF9b dimer binds to the membrane with a β-folds rich conformation,involving mature virion assembly,which implicates the maximized use of the viral genome.Avian influenza clades and subclades of H5Ny have been reported to cause human infections,especially clade 2.3.4,which includes the H5N1 and H5N6 subtypes.The most recent 2020-2021 H5N8 outbreak has affected poultry and wild birds worldwide.In November 2020,an outbreak of H5N8 avian influenza virus infection in whooper swans occurred in the Yellow River Reservoir region of China,and several deaths were found.On 18 February 2021,seven cases of human infection with H5N8 were reported in Russia.The continuous evolution of H5Ny and its recombination with other subtypes of IAVs deserve attention.What is the molecular mechanism behind the continuous sporadic human infection of H5Ny?To figure out this question,we carry out the second part of this thesis.In this study,we evaluate the receptor binding properties and molecular basis of two H5N8 isolates:A/Astrakhan/3212/2020/H5N8 from Russia(human H5N8)and A/whooper swan/Henan/CAS001-K/2020|H5N8 from whooper swans in China Yellow River reservoir(swan H5N8).Phylogenetic tree analysis showed that the two strains from different sources both belonged to the 2.3.4.4b H5N8 subclade and were closely related.By means of surface plasmon resonance(SPR)and ELISA,we found that the two H5N8 strains preferentially bind to avian receptors with negligible binding to the human receptor,and the molecular mechanism of H5 binding to receptor analogues was elucidated by X-ray crystal structures.In addition,we found that although H5N8 HA cannot bind to human sialic acid receptors,it has evolved the ability to bind to more types of α2-3 sialic acid receptors like Lewis X through glycan microarray.Immunofluorescence staining of tissue sections shows that H5N8 could bind to human trachea.SPR assay also showed that the affinity of H5N8 HA to Sialyl Lewis X sugar was significantly higher than that of the early H5Ny HA.Finally,the complex structure of H5N8 HA with Sialyl Lewis X sugar receptor was resolved by single particle cryo-electron microscopy,which clarified the molecular mechanism of the enhanced affinity between H5N8 HA and Sialyl Lewis X sugar receptor and identified the key sites.This study elucidated a new mechanism for influenza virus to infect humans by binding to more types of α2-3 sialic acid receptors during evolution,which provided a new indicator for the monitor of influenza viruses.In summary,this thesis focuses on the infection and pathogenesis of SARS-CoV-2 and influenza virus.We determined the homodimer structure of SARS-CoV-2 ORF9b with lipid ligands,and identified that the ligand are monoglycerides.We also studied the effect of phosphorylation site on protein stability,and proposed that ORF9b is a fold-switching protein,inndicating that SARS-CoV-2 uses very clever and economical ways to function throughout the life cycle.We found that H5N8 viruses still preferred to bind to avian-derived sialic acid receptors,and K222Q evolution on HA significantly enhanced its affinity with Sialyl Lewis X sugar,which provided new ideas for the prevention and control of avian influenza viruses.