Study On Special PRRSV Binding Amino Acids Of Sialoadhesin Receptor And Their Role On PRRSV Special Infection

PRRS (Porcine reproductive and respiratory syndrome)disease is caused by PRRSV (Porcine reproductive and respiratory syndrome virus)infection worldwide. Infection of PRRSV for pig leads to reduce reproduction, low immunity, high mortality. It causes meat product safety, environment and serious problems.The mechanism for PRRSV infection immunity and immunity restraint is very complicate and unclear. The traditional vaccine and drug is ineffective for PRRSV. The PRRSV has a strong host tropism which can infect domestic and wild pigs but not infect other animals. PAM (Porcine Alveolar Macrophage) cells are main host cell in pigs, and CD163and CD169(SN, Sialoadhesin) are as main receptor molecules on PAM cells. pSN (Pig SN) receptor adheres to sialic acid molecules on the surface of virus, which mediates the virus-infected cells. Some sites on receptor directly affect receptor binding activity of PRRSV, so it is a direction for mechanism research on infection immunity and immunity restraint.Interaction of receptor and PRRSV is the novel idea of this paper to analysis specificity of pSN molecule, which is an important amino acid sites for PRRSV infection tropism. Therefore, the receptor resistant mechanism is the main focus of this study which is also an important molecular target and reference of drug design, genetics and breeding subject.1. Predicted PRRSV binding site on SN based on bioinformatics analysis(1) According to the mSN (mouse SN) PDB database, SN protein templates and sialic acid ligands structure of the template structure were superposed and compared to critical amino acid within6.5A. The results showed that the sialic acid molecule derivatives had great variation, but the mouse sialic acid molecule has a conservative Neu5Ac mother nucleus. In PDB (Protein Data Bank) template, the position of the mother nucleus and the formation of hydrogen bonding interactions of amino acids is also conservative. (2)According to pig SN gene cDNA and protein amino acids sequence, pSN signal peptide cutting sites were predicted on Signal IP and Smart protein structure domain websites. Referring the PDB on mSN template structure, pSN signal peptide cutting sites might be were between amino acids19-20sites.(3)By Smart protein domain prediction, pSN and virus sialic acid-binding region was within the N-terminal,150amino acid region on pSN.(4)Glycosylation prediction has showed on pSN S107, T3, T78etc. Glycosylation may only happen during PRRSV infection.(5)Based on the results of pig and mouse multiple sequence alignment and structure superimposition result. R97, R105amino acid in PRRSV binding process, they may change of side chain direction and hydrogen. And, R106, R2amino acid, with huge side chain as hindrance. They could limit binding direction during the PRRSV sialic acid nucleus when sialic acid combined with SN.(6)Based on multiple sequence alignment, homology modeling and molecular dynamics optimization steps, pSN and cSN (cow SN) protein structure homology models were built. pSN and cSN were superposed with mSN template. SN some of amino acid mutants were built using chimera software for observation. The results determined a cavity could be formed on the surface of pSN and it seems that cavity was not on the surface of mSN and cSN. When S107V on pSN was replaced for V107, cavity became small. On the contrary, it would improve more space for interaction. So,S107was very key site for PRRSV binding.(7)To observe the structure specificity among pSN, mSN and cSN, their surface of107was a specific hydrophilic amino acid, in the process of binding the virus sialic acid. It was possible to provide a hydrogen bond and a specific hydrophilic region to accommodate the sialic acid of the virus. Thereby, it would increase the binding capacity of the virus and pSN. Perhaps, this was the difference in sensitivity and resistant reason between species. Moreover, other amino acid sites of the pig2,44,45,97,105,106,109, etc., were also involved in the formation of the hydrophilic region. 2. Validated key PRRSV binding sites on SN and their function using experiments(1) In order to analyze3,78and107sites role on SN interaction with PRRSV in bioinformatics, we used and site-directed technology the pEGFP-Nl vector for expression SN and GFP fusion protein in293T cells. According to pSN and cow cSN receptor (cSN not binding with PRRSV sialic acid) sequence, results showed2kinds of wild type pSN-GFP, cSN-GFP and4kinds of mutant type pSN-S107V-GFP, pSN-T3V-GFP, pSN-T78V-GFP, SN-V3T-V78T-V107S-GFP.(2) By pEGFP-N1vector, SN-GFP expression vector were built.6kinds of high dose of chimera protein were expressed in293T cells and secreted into cell medium and abstracted by ultra filtration and centrifugation. They are pSN-GFP, cSN-GFP, pSN-S107V-GFP, pSN-T3V-GFP, pSN-T78V-GFP and cSN-V3T-V78T-V107S-GFP, and can be used in molecular level of examination about binding activity of SN and PRRSV.(3) In order to study the SN and PRRSV interaction on cell level, an M transmembrane domain was constructed into SN-GFP vector, so that expression of the vector SN-GFP-M were performed including M transmembrane fragments. They were pSN-S107V-GFP-M, pSN-T3V-GFP-M, pSN-T78V-GFP-M, CSN-V3T-V78T-V107S-GFP-M, pSN-GFP-M, cSN-GFP-M and GFP-M control.(4) Total6kinds of SN-GFP and7kinds of SN-GFP-M target protein were detected by anti-GFP antibody on WB (Western Blot.)(5) Using for Far-Western Blot and ELISA (En2yme Linked Immunosorbent Assay) method, SN wild type protein and the mutant type were detected in vitro interaction with the PRRSV virus including2kinds of wild type pSN-GFP, cSN-GFP and4kinds of mutant type pSN-S107V-GFP, pSN-T3V-GFP, pSN-T78V-GFP and cSN-V3T-V78T-V107S-GFP. The result shown that the binding activity of pSN wild type was better than that of other5kinds of SN-GFP chimera protein. Mutant type pSN-S107V-GFP, pSN-T3V-GFP, pSN-T78V-GFP binding activity were reduced, especially on pSN-S107V-GFP. cSN wild type could not bind with virus well, but another mutant type cSN-V3T-V78T-V107S-GFP also were bind with little PRRSV. (6)Immunofluorescence experiments for PRRSV and SN-GFP-M interaction also were studied. The6kinds of SN-GFP-M vector and a contrast GFP-M containing the SN transmembrane M segments were transfected and expressed into293T cells to examine the binding ability with PRRSV by Immunofluorescence experiments. The pig and cow SN wild type chimera protein,3kinds of pSN mutant type, a cSN mutant type, and a contrast GFP-M were performed binding activity examination. The result was similar with FAR-WB.(7) The above experimental results showed that pSN sites of T3、T78and S107were of great importance for binding with PRRSV, especially S107.(8) The pSN wild-type and the PRRSV virus had a strong binding ability. In our experiment, some amino acids were replaced from pig to cow in pSN. Pig mutant type pSN-T78V, pSN-T3V especially pSN-S107V reduced binding ability of the virus. But, cow cSN of the107replaced for pig as cSN-V107S, it could restore part of the binding ability of the virus from bioinformatics analysis. The mutant type cSN-V3T-V78T-V107S-GFP also could bind with some PRRSV. So, it could be inferred that these key sites are very important and special for resistance in different breeds of pig and other species infection by PRRSV.