Investigation Of The Function Of Non-muscle Myosin Heavy Chain Ⅱ-A In PRRSV Infection Of Cells

Porcine reproductive and respiratory syndrome (Porcine Reproductive and RespiratorySyndrome, PRRS) is a contagious disease caused by porcine reproductive and respiratorysyndrome virus (PRRSV). This disease is also called “Blue Ear”. PRRSV mainly causedreproductive disorder in sows and respiratory problems in pigs of all ages and decreased inaverage weight gain. The virus also infected pregnant sows causing immnosuppresion andpersistent infection. PRRSV shares a very narrow host tropism and a marked preference forcells of the monocyte/macrophage lineage. More specifically, in vivo, PRRSV infectssubpopulations of differentiated macrophages, with alveolar marcophages being the majortarget cells during acute infection. In vitro, PRRSV replicates in primary cultures of alveolarmarcophages and to some extent in peripheral blood monocytes. Furthermaore, the Africangreen monkey kidney cells MA104and cells derived Marc-145are showed to sustain PRRSVinfection. Both in PAM and in the monkey kidney-derived cell lines, the virus enters througha mechanism of receptor-mediated endocytosis. The first step in this entry process is theattachment to one or more cellular receptors. Up to now, five mediators have been reported:sialoadhesin (Sn), Heparan sulphate (HS), Vimentin, CD163and CD151. HS can mediatevirus attachment but no internalization; Sn is sufficient for both PRRSV attachment andinternalization; CD163maybe play a role in viral uncoating and genomic RNA releasing. Theinteraction of NMHC-ⅡA and PRRSV was verified by Mab2-5G2which mimicked the GP5antigen inhibited the interaction between idiotypic anti-GP5antibodies and GP5antigen. Thisprotein is about230KD and it maybe a new receptor for PRRSV. Using mass spectrometricanalysis and sequencing, we kown it is non muscle myosinⅡA. In mammals, three differentisoforms of nonmuscle myosinⅡ,Ⅱ-A,Ⅱ-B,Ⅱ-C, are widely distributed throughout the entireorganism. These proteins play a role in many fundamental cellular and development processessuch as cell-cell adhesion, cell migration and cytokinesis. Up to now, the researches ofNMHC-ⅡA are mainly focus on the role of it playing in the cells. There is rare study on theinteraction between the NMHC-ⅡA and virus. Arii et al.(2010) showed that nonmusclemyosin heavy chain ⅡA (NMHC-ⅡA), a subunit of nonmuscle myosin ⅡA (NM-ⅡA),functions as a herpes simplex virus-1(HSV-1) entry receptor by interacting with glycoproteinB. The soluble CD163was verified to associate with non-muscle myosin heavy chain type Ain T lymphocytes. Whether the CD163interacted with NMHC-ⅡA in Marc-145cells and hadrelationship with PRRSV infection was still not confirmed. In this study, we focus on theinterrelationship between NMHC-ⅡA and PRRSV to do some analysis as following: 1. The interaction of NMHC-Ⅱ A protein with PRRSV virus particles invitroIn order to analysis the interrelationship between NMHC-ⅡA and PRRSVunambiguously, we process the co-immunoprecipitation method to extract NMHC-ⅡA proteinfrom Marc-145cell, and prokaryotic expression the truncated NMHC-ⅡA protein PRA. Weusing the Protein-Virus pull down method to analysis the interaction of PRRSV with proteins.The Western blot result showed that PRRSV can be captured by proteins, while control wasnegative.2. The virus infection blocking by anti-NMHC-ⅡA serumAs the mediators of PRRSV infection of host cells CD163, Vimentin, Sn, HS and CD151,The transfection of those protein expression clone into unsusceptible cell could render thesecells susceptible to PRRSV infection, and the transfection of siRNA against those protein intoMarc-145significantly reduced the level of PRRSV infection. Also, anti-those proteinsantibody treatment to Marc-145completely blocked PRRSV infection. In order to verifywhether the potential mediator NMHC-ⅡA could also block PRRSV infection, we used theNMHC-ⅡA protein purified by co-immunoprecipitation method to immunized mouse, andblocked the PRRSV infection with mouse serum. The result displayed that1:5dilution serumcould block80%PRRSV infection and with the dilution extension the block efficiency wasdown.3. NMHC-ⅡA knock down stable cell line establishment and virus infectionassay.To evaluate the function of NMHC-ⅡA in PRRSV infection to Marc-145cells, RNAinterference (RNAi) approach by amiRNA was performed to specifically silence theNMHC-ⅡA. We used four amiRNA sequences directed against NMHC-ⅡA for their capacityto down-regulate NMHC-ⅡA expression (miRNA-1, miRNA-2, miRNA-3, and miRNA-4), aswell as one control sequence. Five gene sequences were synthesized and cloned intopcDNATM6.2-GW/EmGFPmiR vector to construct plasmids. Five plasmids were transfectedinto Marc-145cells and the transcient transfection cells were assessed by RT-PCR, theRT-PCR detection result displayed that miRNA-4transfected cell could knock downNMHC-ⅡA expression more efficiently. So miRAN-4and miRNA-NC transfected cells P-4and P-NC were selected by subclone. Through the RT-PCR result, the NMHC-ⅡA expressionlevel of P-4was down regulation47%compared with P-NC. PRRSV infection result showedthat47%down regulation of NMHC-ⅡA expression could not affected the virus binding to cells, but the propagation titration of PRRSV was decreased, it was more obviously at24hpost virus infection, the percentage of decreasing titration were about42.1%，22.3%，13.2%and12.6%at24,48,72and96h post virus infection. It could be confirmed that downregulation NMHC-ⅡA expression could decrease PRRSV infection.4. Analysis of co-localization of NMHC-Ⅱ A protein with PRRSV inMarc-145cellsIn order to more intuitively observe the interaction of NMHC-ⅡA and PRRSV duringvirus internalization, we fixed the P-4, P-NC and Marc-145cells at different time afterPRRSV binding to cells, and stain virus and NMHC-ⅡA with different fluorescence thenobserve the co-location of them. The result demonstrate that the virus absorption to three cellshave no significant different, but the virus internalization amount of P-4cells was moreobviously decreased compared with P-NC cells. And the time of virus internalization on P-4cell was delayed15min compared with control cells. Another point the virus internalizationprocess was accompanied with NMHC-ⅡA contraction, and the time of Virus moved from thecell membrane into the cytoplasm and forward to nucleus was a relatively short time, andafter arriving at a certain distance around the nucleus and the relative position within a certaintime no longer changes.5. NMHC-ⅡA protein knock down cell growth curve measure, cell cycledetermination and mitosis observationIt was reported that NMⅡ takes centre stage in cell adhesion, migration and cytokinesis. Inorder to analysis whether the down regulation of47%NMHC-ⅡA would affect the celldivision and proliferation, and then affect the virus infection, the cell proliferation curve, cellcycle measure and cytokinesis observation were performed. The assay result confirmed thatthere was no significant different between47%down regulation of NMHC-ⅡA cells andcontrol cells in cells proliferation, and the percentage of cell cycle distribution of three cellschanges was consistent, and cell cytokinesis was normal.