Association Of CD163 With Other Proteins In PRRSV Infection Of Cells

Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) is a contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). This disease is also called"Blue Ear". PRRSV mainly caused reproductive disorder in sows and respiratory problems in pigs of all ages and decreased in average weight gain. The virus also infected pregnant sows causing immnosuppresion and persistent infection. PRRSV shares a very narrow host tropism and a marked preference for cells of the monocyte/macrophage lineage. More specifically, in vivo, PRRSV infects subpopulations of differentiated macrophages, with alveolar marcophages being the major target cells during acute infection. In vitro, PRRSV replicates in primary cultures of alveolar marcophages and to some extent in peripheral blood monocytes. Furthermaore, the African green monkey kidney cells MA104 and cells derived Marc-145 are showed to sustain PRRSV infection. Both in PAM and in the monkey kidney-derived cell lines, the virus enters through a mechanism of receptor-mediated endocytosis. The first step in this entry process is the attachment to one or more cellular receptors. Until now, four receptors have been reported: sialoadhesin (Sn), Heparan sulphate (HS), Vimentin and CD163. HS can mediate virus attachment but no internalization; Sn is sufficient for both PRRSV attachment and internalization; CD163 maybe play a role in viral uncoating and genomic RNA releasing. Our supervisor immunoprecipitated a protein from MA-104 and PAM cell lysates by Mab2-5G2. Mab2-5G2 represents internal image anti-idiotope which mimicked the GP5 antigen inhibited the interaction between idiotypic anti-GP5 antibodies and GP5 antigen. This protein is about 230KD and it maybe a new receptor for PRRSV. Using mass spectrometric analysis and sequencing, we kown it is non muscle myosinⅡA. In mammals, three different isoforms of nonmuscle myosinⅡ,Ⅱ-A,Ⅱ-B,Ⅱ-C, are widely distributed throughout the entire organism. These proteins play a role in many fundamental cellular and development processes such as cell-cell adhesion, cell migration and cytokinesis. Soluble CD163 with activated T lymphocytes associates with cellular non-muscle myosin heavy chain type A. But so far, the viral proteins involved in the interaction with CD163 are unkown. And the counterpart on the CD163, which binds to the viral protein, is also unclear. The interaction of recetpor to receptor or CD163 to non-muscle myosin is also known litter. The aim of the present study is to investigate the interaction between CD163 and viral protein or non-muscle myosinⅡA/other PRRSV receptor.In order to perform the function of CD163 for PRRSV receptor or the relationship between CD163 and non-muscel myosin, the full length CD163 gene was generated by RT-PCR from porcine alveolar marcrophages (PAM) and Marc-145 cells. The gene was inserted into a eukaryotic expressional vector pcDNA3.1/V5-His A to yield recombinant plasmid pCD163 and mCD163. PK-15 and BHK-21 cells are PRRSV non permissive cell line. They were transfected by purified plasmid (pCD163, mCD163). Then the cells were exposured to PRRSV-12# strain for the infection of virus after 24h post transfection. In IFA test, the PRRSV infected cells could be detectable by anti-N monoclonal antibody. The result was that the PK-15/BHK-21 cells transfected CD163 could be infected by PRRSV. And the same time, It was found that transfection efficiency differed between cell lines, with 10% and 30% transfected cells for BHK-21 and PK-15 cells, espectively. On this base, non-permissive BHK-21 cells transiently expressing recombinant CD163 and PRA (It is sited on the carboxyl-terminal of non-muscle myosinⅡA ), either separartely or combined, were inoculated with the American prototype PRRSV-12# strain and analysed for expression of viral nucleocapsid protein and production of infectious virus. Immunofluorescence experiments showed that the cells were infected. In contrast, when CD163 and PRA were combined, clearly more infected cells were observed. This result showed that PRA can cooperate with CD163 for PRRSV infection. In order to prove the relationship of non-muscle myosinⅡA and PRRSV, The Marc-145 cells were infected by PRRSV at different time to be fixed. Confocal microscopical analysis of PRRSV during infection showed that PRRSV mainly attached the surface of cells at 4℃. When the cells were shifted to 37℃, the virus entered into the cells after about 5min. As time goes on, A majority of PRRSV entered into cells and non-muscle myosinⅡA (NMHCⅡA) became more and more. After 60 minutes, PRRSV entered cells completely, NMHCⅡA also became decreasing. This experiment illustrated that NMHCⅡA is associated with PRRSV infection.Predict the structure of protein and the the functional domain using the Scanprosite softwore. CD163 is a typeⅠmembrance protein composed of a signal peptide by followed by nine SRCR domain, with a thirty-five amino acid proline-serine-threonine (PST) rich region separating SRCR 6 and 7. A second PST rich region connects SRCR 9 with the transmembrane domain and a short cytoplasmic tail, which contains a functional internalization motif. For PAM, full length sequence is 1115 amino acid (AA). The site of SRCR from 1 to 9: 54AA-151AA, 161AA-258AA, 268AA-365AA, 375AA-472AA, 480AA-577AA, 585AA-682AA, 721AA-818AA, 828AA-925AA and 931AA-1028AA, seperatively. The first PST is located on 683AA-720AA, the second PST and the cytoplasmic tail is between 1029AA and 1115AA. For Marc-145 cells, there is no report about its full length sequence. According to the high homology of CD163 from PAM and Vero cells, the primer was designed and amplified the full length sequence by RT-PCR. The gene is 1117 amino acid and was registered in GenBank. The GenBank accession number was JF53553. Its SRCR 1 to 9 is located on 54AA-152AA, 162AA-259AA, 269AA-366AA, 376AA-473AA, 481AA-578AA, 586AA-683AA, 722AA-819AA, 831AA-926AA and 932AA-1029AA, seperatively on the full length sequence. The first PST was located on 684AA-721AA, the second PST and the cytoplasmic tail was located on 1030AA-1116AA. The signal peptide is from initiation codon to 16AA, it guides the transmembrane of CD163.According to the structure of CD163 and the related references, subsequently, the full length of CD163 from PAM and Marc-145 cells was dicided into a series of fragments. The fragments from PAM were named P1, P2 and P3, seperatively; the fragments from Marc-145 cells were called M1, M2 and M3, seperatively. These fragments were cloned into pMD18-T vector, and then inserted into eukaryotic expressional vector pcDNA3.1/V5-His or pEGFP-N1. These fragments were named Z-P1, Z-P2, Z-P3, Z-M1, Z-M2 and Z-M3, seperatively. Z-P1 was 798 base pair (bp) and lacated on 1-798bp, Z-P2 (1257bp) was between 790bp and 2046bp, Z-P3 (1323bp) was 2023bp-3345bp; For Z-M1, Z-M2 and Z-M3, It was lactaed on 1bp-777bp, 763bp-2049bp and 2035bp-3351bp. The length is 777bp, 1287bp and 1317bp, seperatively. These positive plasmids were transfected into non-permissive cells BHK-21 and PK-15 for the viral infecting test. The results were that P3 and M3 were high transfection efficiency compare to other fragments. P1 and M1 were in the last place, and it was found that the transfection efficiency was higher in BHK-21 than PK-15. The transfected cells were exposured to PRRSV-12# strain. In IFA test, the PRRSV could be detectable by anti-N monoclonal antibody. The experiment showed that P1 and M1 could not been infected by PRRSV and other plasmids could do. The infected degree of P2 and M2 was stronger than P3 and M3. These results illustrated that SRCR 1-2 were not required for PRRSV infection. SRCR 3-6 were involved in PRRSV infection.Make further investigation of CD163 for PRRSV infection /PRRSV receptors or receptor to viral proeins and receptor to NMHCⅡA. CD163 fragments were expressed into prokaryotic vector pET-28a (+). CD163 gene included many rare codons by the Graphical codon analysis. The P1, M1, P3 and M3 fragments were truncated again. The new fragments were named Y-P1, Y-M1, Y-P3 and Y-M3, seperatively. They were 639bp, 618bp, 942bp and 978bp and the sequence coded the amino acid from 160-798bp, 160bp-777bp, 2143bp-3084bp and 2143bp-3120bp. The new CD163 fragemts were correct by sequencing. The plasmids were transformed into Rosetta competent cell and induced by IPTG at different conditions. The recombinant proteins were about 24KD, 46KD, 35KD, 22KD, 46KD and 36KD by SDS-PAGE. Western blot test confirmed that the expressed recombinant protein could specifically react with the anti His-Tag mouse monoclonal antibody. The furified recombinant protein was immunized Balb/c mouse and obtained the hyper-immune serum. Y-P3 and Y-M3 mouse serum could block PRRSV infection Marc-145 cells at 1/20 or 1/40 dilution by the serum block assay, while Y-P2 and Y-M2 could improve the viral infection. For Y-P1 and Y-M1, there is no obvious difference compare to the positive control. For the protein binding cells experiment, CD163 fragment protein chould bind Marc-145 cells in IFA. P3 and M3 proteins bound weaker than other proteins at 200μg/ml concentration. This result clarified that CD163 fragments maybe bind some proteins on the surface of Marc-145 cells. M1 and M3 proteins could bind NMHCⅡA by co-immunoprecipitation and Western Blot. For ELISA test, GP5 protein could not bind CD163 fragments. For GP5 fragments protein (GP5-1, GP5-2, GP5-3, GP5-4), GP5-1 could bind P1, P2, M1 and M3; GP5-2 bound P1, P3 and M1; GP5-4 was interaction with P1 and M1, while GP5-3 could not do with CD163 fragments. For Sn fragmenrs Sn-1 and Sn-3, Sn-1 could bind P1, M1 and M3 and Sn-3 was interaction with M1 and M3.