The Researches On Receptor Binding Characteristics Of The S Protein And Structure Of The NSP9 Protein Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus(PEDV) is a member of Alphacoronavirus, it can infect piglets of all ages. Recently, the outbreak of porcine epidemic diarrhea(PED) has caused huge economic losses for the global pork industry. The genome of PEDV can encode 16 non-structural proteins(NSP1-NSP16), ORF3, S, M, E, N protein, each protein plays an important role in virus life cycle. PEDV S protein is a membrane glycoprotein on virus surface, it plays a crucial role in regulating the interaction with its receptor protein on host cell surface, the epitope and receptor binding domain can be a target for vaccine and antiviral drugs development. NSP9 protein is characterized by nucleotide binding activity, it is a member of viral replication complex and plays an important role in viral replication. This study used PEDV S protein and NSP9 protein as the research object, explored the receptor usage characteristics of PEDV S protein and the structure and oligonucleotide binding characteristics of NSP9 protein, the detailed contents are shown as follows:1. In this study, the receptor usage characteristics between PEDV S protein and its protein receptor pAPN or co-receptor sugars were studied. To identify the receptor binding domain in the PEDV S protein, five different truncated fragments of S1 protein and pAPN protein were constructed, the results of flow cytometry and ELISA assays showed that the RBD of PEDV located in the C-terminal domain of the S1 protein(residues 253-638), the receptor binding domain is broader than other coronavirus. Sequence of the CHGD-01 S253-638 fragment and the CV777 S249-634 fragment exhibited very high similarity, the results of flow cytometry showed that they exhibited similar pAPN-binding activity, indicating that the prototype strain owns similar receptor-binding activity with the variant field isolate. PEDV S1 C-terminus(S477-629) showed similar structure and sequence as TGEV, HCoV-NL63 receptor binding domain, thus we infer that the three loops at the tips of the β-barrel domains of PEDV S1 C-terminus may be responsible for receptor binding, therefore, the three loops from the CHGD-01 S477-629 fragment were individually replaced with those of HCoV-NL63 or the sequence “SGSGS”, the ELISA, dot-blot hybridization, and pull-down assay results showed that they did not completely lose their pAPN-binding activities, indicating that the predicted three loops did not play a crucial role in the PEDV S-pAPN association, thus, PEDV conforms to a different receptor recognition model compared with TGEV, PRCV, and HCoV-NL63. To determine whether the PEDV S protein possesses sugar-binding activity, full-length S1 and seven truncated S1 fragments were constructed, the results of ELISA assay demonstrated that the N-terminal region of the PEDV S1 domain possesses sugar-binding activity. Meanwhile, the CHGD-01 S1-324 fragment showed stronger mucin binding activity than the CV777 S1-320 fragment, indicating that the sugar-binding activity of the PEDV variant field isolate was stronger than that of the prototype strain.2. The structure and oligonucleotide binding characteristics of NSP9 protein were studied. After the process of protein expression and purification, crystal screening and optimization, the crystal structure of NSP9 protein was analyzed, the structure showed that NSP9 protein monomer contained 7 β-strands and a α-helix appended at the C terminus. Crystal structure of NSP9 protein revealed two different dimers: One of the dimer was formed by the parallel association of the C-terminal α-helices, amino acids Gly95, Gly99 and Gly102 formed the main hydrophobic interaction; another dimer was formed by the disulfide bond and hydrogen bonds, hydrophobic interactions in β-strand 4 and 5. Crosslinking and AUC assays results demonstrated that monomers and dimers were the main form for NSP9 protein in solution. SDS-PAGE and AUC results have confirmed the monomeric state of the NSP9C59 A mutant, whereas the NSP9G95 E,G99E,G102 E mutant showed the same state as the wild type NSP9, which demonstrated the pivotal role of disulfide bond in NSP9 dimer formation, the analyzed crystal structure of the NSP9C59 A mutant further confirmed the above results. Analyzing has found that many positive charged residues distributed on the surface of PEDV nsp9 structure, strong positive potential present on the hydrophobic dimer interface or in the central domain of each monomer, indicating it is an accessible surface for nucleic acids interaction. To test whether the amino acids Cys59, Gly95, Gly99, Gly102, Lys10、Arg68、Lys69、Arg106 are related to nucleic acid binding, a series of related mutants were constructed, the EMSA and MST assay results showed that the critical amino acids Cys59, Gly95, Gly99, Gly102 that participating in dimer formation didn’t have obvious effect on nucleic acid binding, but the positively charged residues of NSP9 protein surface played important role in nucleic acid binding, meanwhile, the apparent affinity of PEDV NSP9 protein did not depend on the ssDNA length.The structure and function of PEDV S protein and NSP9 protein exhibited different characteristics with other coronavirus, thus, PEDV owns its unique invasion, replication, and pathogenic mechanism. This study provided the theoretical foundation for elucidating PEDV pathogenesis and new idea for developing vaccines or antiviral drugs.