Identification Of Linear B Cell Epitopes On PRRSV M Protein And Its Interaction With Host Protein

Porcine reproductive and respiratory syndrome virus (PRRSV) is regarded as an important pathogen impacting on the swine industry worldwide. The M protein, the most conserved structural protein of PRRSV, is unglycosylated. In the virion, the GP5and M proteins are found as a disulphide-linked heterodimer, which is essential for PRRSV infectivity. In the present study, the monoclonal antibodies (MAbs) M protein were prepared, and the corresponding epitopes were identified, and then the host cellular proteins that interact with the M protein were screened, finally, the molecular mechanism of the interaction of M protein with the host cellular protein NF45and the role of their interaction in the PRRSV replication were explored. The objective of this study is to provide scientific evidence for elucidating the biological fuctions of M protein during the PRRSV infection and immunity.The MAbs against PRRSV HuN4-F112were obtained by fusing myeloma cells SP2/0and spleen cells of six week-old BALB/c mice immunized with ultracentrifugation-purified HuN4-F112in this study. Two hybridoma cell lines against HuN4-F112M protein were prepared by screening with the immunofluorescence assay (IFA) and subcloning, named1C8and3F7, respectively. The two MAbs with IFA titers of1:512were identified to be IgG2a and IgGl, respectively. Western blot analysis showed that the two MAbs could specifically recognize the M protein of PRRSV. Blocking ELISA showed that the binding of the1C8and3F7to PRRSV could be inhibited by the anti-serum to PRRSV.The epitopes on M protein were characterized using the two MAbs. The ORF6gene was divided into four overlapping segments for expression. Western blot analysis showed that MF1(aal-55) reacted with MAb1C8, while MF4(aa121-174) reacted with MAb3F7. These two segments were then further divided into eight fragments. Upon identification of bound polypeptide, complementary oligonucleotide pairs were synthesized, annealed together, and cloned into the expression vectors. The results showed that1C8and3F7recognized the minimal epitopes aa3-6(3SSLD6) and aal55-163(155VLGGRKAVK163), respectively. The two epitopes recognized by MAbs1C8and3F7were considered to be novel linear epitopes on M protein. Alignments of epitope amino acid sequences revealed that the two epitopes on M protein were highly conserved among the genotype2PRRSV strains, while deletions and mutations existed in the genotype1strains.The host cellular proteins interacting with the M protein of PRRSV were immunoprecipitated from MARC-145cells infected with HuN4-Fl12by the MAb3F7and then identified by LC-MS/MS. The screened proteins were used for bioinformatics analysis. The results showed that PRRSV HuN4-F112infection group had10differentially expressed protein bands compared to control group. Totally,285cellular proteins interacting with M protein were identified by LC-MS/MS, which are involving in the cellular pathways associated with protein translation, infectious disease, and signal transduction. HEK293T cells were cotransfected with the eukaryotic expression vectors carrying HuN4-F112-M and NF45ORFs, and the interaction and colocalization between PRRSV M protein and NF45were verified by using co-immunoprecipitation and confocal microscopy techniques. MARC-145cells were infected with PRRSV HuN4-F112, and NF45could be detected in the immune complex after co-immunoprecipitation using the MAb3F7, indicating that the two proteins could interact under the PRRSV infection.MARC-145cells were infected with PRRSV HuN4-F112after the NF45gene was silenced, and the effect of the interaction between NF45and M on PRRSV proliferation were analyzed using Western blot and viral titer assay. The results indicated that the viral titer and the expression of PRRSV M protein decreased significantly after the NF45was silenced in MARC-145cells, suggesting that NF45is involved in PRRSV replication process.In summary, two novel epitopes (aa3-6and aa155-163) on M protein were identified, and the host cellular proteins interacting with the M protein of PRRSV were screened, and the interactome profile was drawn, and the interaction between PRRSV M protein and NF45were confirmed, and the biological significance of their interaction were explored. The findings enrich for understanding the antigen structure of M protein and provide scientific clues for exploring the molecular mechanism and biological function of the interaction of PRRSV with host cellular proteins.