Investigation Of The Interation Between PRRSV Anti-idiotype Antibody Mab2-5G2 And MYH9

Porcine reproductive and respiratory syndrome virus?PRRSV?is the causative agent of porcine reproductive and respiratory syndrome?PRRS?that can result in reproductive failure in sows,respiratory distress in nursing pigs,and poor growth performance during infection.At present,PRRS is endemic and can not be effectively controled and eliminated.In particular,the highly pathogenic PRRSV?HP-PRRSV?outbreak in 2006 caused significant losses for the swine industry in China.Unfortunately,current strategies for controlling PRRSV have been largely inadequate,since new PRRSV variants are constantly emerging.Auto-anti-idiotypic antibodies?Aab-2s?were detected from pigs infected with PRRSV.Aab-2s specific against the idiotypic antibodies?Ab-1s?to the envelope glycoprotein GP5 or M protein of PRRSVwere produced,including Aab-2s could served as internal image of GP5protein.Anti-idiotypic antibody?Ab-2?could be used as a vaccine candidate and served as a tool to screen and identify viurs receptor from susceptible cells.Our previous study has demonstrated that monoclonal Ab2 against antibody to PRRSV GP5,designated Mab2-5G2,recognizes PRRSV permissive cells by interacting with MYH9from PAMs or MARC-145 cells,which is a novel cellular factor that binds to PRRSV major glycoprotein GP5.Based on these studies,the objectives of this research:1.Identification of the key domain of MYH9 interaction with Mab2-5G2Non-muscle myosin II?NM II?plays important roles in the broad ranges of biological functions,such as cell migration,mitosis,vesicle metastasis repair,cytokinesis and secretion.NM II family contains three isoforms,NM II-A?MYH9?,NM II-B?MYH10?,and NM II-C?MYH14?.The amino acid homology among them is 64%to 80%.The differences between them were located at the C terminus.In order to find the binding domain of Mab2-5G2,we first amplify the full length of swine MYH9 using degenerate primers,the full-length were trucated into three fragments,and each fragment were constructed into eukaryotic expression vector pCAGEN-HA,the trucated fragment of MYH9 were transfected into HEK-293T cell for 48 h.The key domain named PRA,for Mab2-5G2 binding,was determined by western blot.2.Recombinant MYH9 Protein C-Terminal Domain?PRA?Blocks PRRSV infectionIn order to test the anti-PRRSV activity of PRA in PRRSV susceptible cells.The recombinant PRA protein was expressed and purified from a prokaryotic expression system.The biological activity of PRA was confirmed using Mab2-5G2 by Western blot and ELISA.Our results show that PRA exhibited a broad-spectrum anti-PRRSV activity.?1?PRRSV SD16 or VR-2332 infection was inhibited by preincubation with PRA in a dose-dependent manner in both susceptible monkey cells?MARC-145?and swine cells(PK-15^CD163,CRL2843^CD163,and PAMs).?2?PRA inhibits both PRRSV-1?GZ11-G1 and P073-3 strains?and PRRSV-2?JXA1,VR-2385,CH-1a,and GD-HD strains?.?3?PRA can capture PRRSV virions via interation with GP5 protein.?4?PRA prevents the membrane redistribution of MYH9 and internalization of PRRSV virions.3.Identification of the MYH9 protein key domain involved in entry of PRRSVPRRSV enters the target cells via receptor-mediated endocytosis,both CD163 and MYH9 are indispensable factors for PRRSV attachment and internalization.However,we do not know whether MYH9 from other species has the ability to mediate PRRSV entry into the target cells,and MYH9 key domain that mediates PRRSV infection.To answer these quentions,we first constructed stable pCD163 expression cell lines from human,murine,and swine origins and then test whether MYH9 from other species could mediate PRRSV infection.Blebbistatin or the small interfering RNA?siRNA?against MYH9s was added to PRRSV susceptible HEK-293T^CD163,PK-15^CD163D163 and BHK-21^CD163 cells.After 48h PRRSV SD16 incubation,PRRSV N gene and proteins were detected using IFA,qPCR and Western blot.Next,PRA from other species was produced in E.coli BL21,the blocking effect on PRRSV infection was detected as above.Further,the crucial common domain of MYH9 from different species for PRRSV binding was determined using inhibition assay of PRRSV infection by the analysis of sequence alignment and PRAs truncation.Finally,the key domain for PRRSV infection was determined at MYH9^1676-1791.Moreover,the mouse polyclonal antibody of MYH9¹676-1791676-1791 could signicantly block PRRSV infection.4.Determination of binding site between Mab2-5G2 and MYH9In order to determine whether Mab2-5G2 could serve as blocking agent for PRRSV infection.The results showed that Mab2-5G2 blocked PRRSV SD16,JXA1,CH-1a,VR-2332,VR-2385 and GD-HD strains infection of PAM.In addition,the binding site between Mab2-5G2 and MYH9 was determined using the ELISA,and Western blot.The spatial structure between Mab2-5G2 and MYH9 was analyed with Homologous Modeling and Docking using Chimera software.The key amino acids in the binding site were predicted at E1670,K1673,E1679 and I1683 in PRA domain.We further produced the recombinant PRA mutants?E1670A,K1673A,E1679A and I1683A?named PRA¹⁶⁷⁰,PRA¹⁶⁷³,PRA¹⁶⁷⁹,PRA¹⁶⁸³,and PRA^{1670,1673,1679,1683},respectively,to test the binding activity to Mab2-5G2 and inhibitory effect on PRRSV infection.The results showed that the interaction between PRA mutants and Mab2-5G2 was significantly redued,the anti-PRRSV activity of PRA mutant was significantly lower than wide type PRA,which suggest that amino acids E1670,K1673,E1679,and I1683 in PRA maybe also invoved in the interaction of MYH9 and PRRSV GP5.Our study indicates that the domain for Mab2-5G2 binding were determined at the C-terminal of MYH9?named PRA?,which exhibited a broad-spectrum anti-PRRSV activity,PRA can capture PRRSV virions via interation with GP5 protein and reduces the membrane redistribution of MYH9 and internalization of PRRSV.MYH9 from other species could mediate PRRSV infection,the key domain for PRRSV infection was determined at MYH91676-1791.Mab2-5G2 could serve as a novel competitive inhibitor for PRRSV entry.Based on homologous modeling of Mab2-5G2 and its interacting domain from MYH9,several key acid residues was identified at E1670,K1673,E1679,and I1683.These information will add valuable information for understanding PRRSV entry as well as developing novel anti-viral strategy against PRRSV infection.