Function Of C-terminus Non-muscle Myosin Heavy Chain â…¡-A Protein In PRRSV Infection Of Marc-145Cells

Porcine reproductive and respiratory syndrome (PRRS), which has prevailed in the main world while pig-raising country and districts, is a contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRS mainly caused reproductive disorder, including of premature, abortion, fetal death, mummy and porcine respiratory syndrome at variety of ages and high mortality. It is also one of the most economically important diseases affecting the swine industry all over the world.After attachment to cellular receptors, PRRSV enters cells by a process of receptor-mediated endocytosis. CD163and vimentin has been identified as receptors for PRRSV on Marc-145cells. CD163is a cellular protein in the scavenger receptor cysteine-rich superfamily, functioned as tripping in the shell and releasing viral genome RNA into the plasma. Vimentin is thought to be involved in the replication and transportation of PRRSV inside the cell by forming a complex with the other components of the intermediate filaments.In the year of2008, Mab2-5G2which is an anti-idiotype monoclonal antibody against GP5antigen of PRRSV identified Nonmuscle myosin heavy chain â…¡-A (NMHC â…¡-A) from PAM and MA-104cells by immunoprecipitation. NMHC â…¡-A is one isoform of NM II heavy chain and plays a role in a variety of cellular processes including cell migration, cytokinesis, cell adhesion and cell shape change both during development and in the adult organism. NMHC â…¡-A was identified as a functional entry receptor for herpes simplex virus-1recently. The C-terminal of NMHC â…¡-A was also identified to block PRRSV infection of Marc-145cells.In order to further study the function of NMHC â…¡-A in PRRSV infection of Marc-145cells, C-terminal of NMHC â…¡-A was selected as the target for searching the binding site of NMHC â…¡-A reacting with Mab2-5G2and verifying the role of the C-terminus NMHC â…¡-A in PRRSV infection of Marc-145cells. To gain the goal, three parts of experiments were taken as follows:Firstly, identify the binding site on NMHC â…¡-A reacting with Mab2-5G2and verify the role of the C-terminal of NMHC â…¡-A in PRRSV infection of Marc-145cells. PRA protein was truncated, deleted, mutated and expressed in E.coli. The binding site on NMHC â…¡-A reacting with Mab2-5G2was identified to locate between aal677to aal713and the key amino acids affecting the reaction were aa1678,aa1682and aa1700. Real time observation was used to detect the effection of the proteins to cell cycle and cell cytokinesis. On the condition that the proteins has no effect on cell cycle and cell cytokinesis, virus neutralization test (VNT) and fluorescence focus neutralization (FFN) were taken to identify the inhibiting function of the proteins to PRRSV infecting of Marc-145cells. Quantitative RT-PCR was used to detect the mRNA level of NMHC â…¡-A and CD163and Western Blot was used to detect the protein level of NMHC II-A,CD163and actin. The results showed that the proteins contain the binding site presenting obvious inhibition to PRRSV infection of Marc-145cells and the proteins contain one or no binding site presenting obvious decrease of inhibition. Besides, the proteins have no effect on mRNA and protein level of NMHC â…¡-A and CD163in Marc-145cells. The results proved that the binding site on NMHC â…¡-A reacting with Mab2-5G2can influence PRRSV infection of Marc-145cells.Secondly, eukaryotic expression of PRA protein in sf9cells and identification of its function in PPRSV infecting Marc-145cell. PRA protein was expressed in sf9cells and purified. PRA protein specifically bound with Marc-145cells and blocked the PRRSV infectivity of Marc-145cells up to60%. These results proved that the structure of the protein can influence PPRSV infection of Marc-145cell, but there were other factors affecting this process, such as NMHC â…¡-A reacting with other receptors, and so on.Finally, identify the interaction of PRA protein with receptors of PRRSV. Immunoprecipitation was used to identify the interaction between PRA protein and proteins in Marc-145cells. The results showed that PRA protein can interact with NMHC â…¡-A and CD163and the truncated proteins of PRA can also interact with NMHC â…¡-A and CD163. The mechanism of the interaction between NMHC â…¡-A and CD163needs further study.All of the results proved that the C-terminal of NMHC â…¡-A influence the infectivity of PRRSV. This work provides a noval approach for further understanding of PRRSV infection and control of PRRS.