Tructural And Functional Studies On CD163 As An Indispensable Receptor Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome(PRRS),is a highly infectious disease in pigs caused by PRRS virus(PRRSV).PRRS is characterized by reproductive failures in pregnant sows,including mummified,stillborn and aborted fetuses,and respiratory distress in swine of all ages.PRRS has widely spread worldwideand become one of the most important infectious diseases,which slaughters and causes huge economic losses to the global pig industry.PRRSV is a single-stranded positive-sense RNA virus with envelope.The virus has the characteristics ofhigh variability,immunosuppression and persistent infection,making it difficult to prevent and control.Therefore,PRRSV has been the focus of veterinary research.PRRSV infectionhas been found to be mediated by an essential receptor CD163.CD163 is an important member of the scavenger receptor family,which is composed of extracellular,transmembrane and cytoplasmic region.Its extracellular domain contains nine scavenger receptor cysteine-rich(SRCR)domains and two proline-serine-threonine(PST)-rich motifs.Its fifth SRCR domain(SRCR5)plays a key role during PRRS infection.However,the structure of CD163 SRCR5 and the important residues involved are not yet clear,hindering the deep understanding of the interaction between PRRSV and CD163,and the virus pathogenesis.Therefore,I carried out structural and functional studies on CD163,which will clarify PRRSV infection mechanism and lay the foundation for the prevention and control of PRRSV with great scientific significances and practical applications.In this thesis,I studied the structures and functions of CD163 as follows:I successfully prepared the recombinant pCD163 SRCR5 using Drosophila melanogaster(S2)cells and Pichia pastoris X-33 and resolved its crystal structures at a high resolution(2.0 and 1.8 ?).The structure includes a distinct long loop region and a special electrostatic potential,which is different from other members of the scavenger receptor superfamily.Subsequently,six singlepoint mutations were performed based on the structure.The PK-15 cells transfected with CD163 R561 A showed a significant production decrease for the classical PRRSV strain BJ-4 and the highly pathogenic PRRSV strain HN07-1(p<0.05).The PK-15 cells transfected with CD163 R561 A further showed a significant decrease in virus binding and entry(p <0.05).All these results identified that the arginine residue at pCD163 position 561 played an important role in PRRSV infection especiallydruing the virus binding to the receptor.This work firstly determined the crystal structure of CD163 SRCR domain and identified an important residue for PRRSV infection,deepening the understanding of PRRSV infection mechanism and providing the molecular basis for the prevention and control of the virus.A recent study shows that pigs lacking functional pCD163 are resistant to type 1 and 2PRRSV,confirming that CD163 is an indispensable receptor for the virus infection.Interestingly,although CD163 SRCR5 was shown to play a key role in PRRSV infection in vitro,gene-edited pigs with pCD163 SRCR5 replaced by a CD163-like homolog(CD163-L1)SRCR8 are resistant to type 1 but not to type 2 PRRSV.Therefore,the mechanism of CD163SRCR5 involved in host cell tropism is not elucidated.In this study,I prepared simian CD163 SRCR5,human CD163 SRCR5 and human CD163-L1 SRCR8 in eukaryotic expression systems.Subsequently,I determined their crystal structures and based on their structural alignments,identified on the specific residues involved in PRRSV infection.All these results may provide some clues of host cell tropism for PRRSV.In this study,I successfully expressed CD163 SRCR5-9 in Drosophila S2 cells and the target protein with high purity through multi-step purification.After primary screen and optimization,I finally obtained diffractive crystals.Subsequent structural and functional assays are being carried out.Because PRRSV has the characteristics of antibody-dependent enhancement(ADE),high degree of antigen variability and so on,there is currently no effective means of PRRS prevention and control.Prevention and control research from PRRSV receptors may be a new idea.Studies have confirmed that pCD163 is an essential receptor for PRRSV infection of host cells,and its fifth SRCR domain is a key domain mediating PRRSV infection.Therefore,this study envisages the preparation of pCD163 SRCR5 domain monoclonal antibodies and screening for specific antibodies that inhibit PRRSV infection.By analyzing the crystal structure and its structure with the receptor complex,a corresponding binding site was found and a small molecule or polypeptide inhibitor was designed to block the binding site of CD163 and PRRSV,thereby inhibiting PRRSV infection.In addition,there is a lack of efficient pCD163 commercial monoclonal antibodies at home and abroad,which brings a lot of inconvenience to the study of pCD163 related functions.The pCD163 SRCR5 domain-specific monoclonal antibody prepared in this study can provide a useful tool for the study of pCD163 function.