| Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the family Arteriviridae in the order Nidovirales. It is the etiologic agent of porcine reproductive and respiratory syndrome (PRRS) which is one of the most important viral diseases threatening the whole world’s swine industry. PRRS mainly leads to reproductive failure such as abortion, fetal death, mummy fetal and so on, in pregnant sows and is involved in the porcine respiratory problems of all ages.PRRSV has a typical cell tropism, which is supposed to be determined by specific cell receptors. This is because PRRSV infection to its host cells occurs via binding to receptors on the surface of cell membrane as a first step and via interacting with cytoplasmic receptors to finish the whole process. The most widely used cell model to investigate PRRSV in vitro is MARC-145 cell line. The main cell receptors involved in PRRSV infection of this cell line includes a heparin-like molecule to which PRRSV binds, vimentin which is suggested to interact with the nucleocapsid of PRRSV followed by transport of the virus to the cytosol, CD151 that may be responsible for fusion of the viral envelope and the endosome, and CD 163 which is essential but unclear for its role in this process.Previously, non-muscle myosin heavy chain Ⅱ-A (NMHC Ⅱ-A) was identified as a potential PRRSV cellular receptor on MARC-145 cells by a monoclonal anti-idiotypic antibody (Mab2-5G2) which functionally mimics PRRSV GP5 protein. It is a subtype of non-muscle myosin heavy chain Ⅱ (NMHC Ⅱ) and is mainly involved in cell migration, adhesion, morphologic change and cytokinesis. It has been reported that NMHC Ⅱ-A mediates signal transduction of chemotactic factor by interacting with CXCR4, and could function as a cellular receptor of herpes simplex virus type Ⅰ. The investigation of this similar function of PRRSV is still in process.The functional domain in NMHC Ⅱ-A has been identified at its C-terminus and the corresponding recombinant protein (designated PRA) was produced. The objective of this study was to determine the role of PRA in PRRSV infection in MARC-145 cells stably expressing exogenous PRA gene. PRA gene was amplified by PCR from pMD18-T-PRA plasmid and cloned into PB donor vector with HA tag sequence in its 5’terminal via restriction enzyme sites Xba I and Not I. Based on sequencing, the desired recombinant plasmid was selected and named PB-CMV-PRA-EFla-GFP-Puro. MARC-145 cells were co-transfected with this plasmid and PB helper vector plasmid and subjected for single cell cloning with puromycin. The presence of PRA gene and its expression in MARC-145 cells were detected by RT-PCR, indirect immunofluorescence assay (IFA) and western blot and MARC-145 cell stably expressing PRA was named MARC-145-PRA. To determine the effect of PRA on PRRSV infection of MARC-145-PRA cells, CPE observation and TCID50 detection were used. The results showed that stably expressing PRA in MARC-145-PRA cells could decrease PRRSV infection. To detect the effect of PRA on PRRSV internalization and attachment, qRT-PCR and confocal laser scanning microscopy were performed. The results showed that stably expressing PRA in MARC-145-PRA cells could inhibit PRRSV PRRSV internalization and attachment as well. Co-immunoprecipitation experiment results showed that PRA could bind with NMHC Ⅱ-A in MARC-145-PRA cells, which was supposed to competitively block the interaction between NMHC Ⅱ-A and CD 163, and this should be responsible for the decrease in PRRSV infection.In summary, the function of C-terminal of non-muscle myosin II heavy chain type A (PRA) in PRRSV infection was studied. One MARC-145 cell line that stably expressing PRA was generated by PiggyBac transposon system and PRRSV infection assay showed that the PRA protein can reduce PRRSV infection, which indicating that the PRA could be used as a potential PRRSV cellular receptor inhibitor. The results generated in this thesis lay a theoretical foundation for the study of PRA protein in PRRSV infection of host cells. |
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