Marc-145Cell Cycle Synchronization And Association Between Its Expression Of PRRSV Receptor Protein And PRRSV Infection

Porcine reproductive and respiratory syndrome (PRRS) is a contagious disease caused byporcine reproductive and respiratory syndrome virus (PRRSV).PRRS mainly causedreproductive disorder, including of premature, abortion, fetal death, mummy and porcinerespiratory syndrome at variety of ages and high mortality. It was discovered in the US in1987and then spreaded in many countries and regions. In1996, the disease was firstidentified in China and has caused the significant losses for the swine industry in China.PRRSVinfectscells through a mechanism of receptor-mediated endocytosis. The first stepof the infection is the attachment of virus to cellular receptors. At present, fourreceptors areidentified which are sialoadhesin (Sn), Heparin sulphate (HS), Vimentin and CD163.We haveidentified non-muscle myosin heavy chain II-A（NMHC II-A）as a new receptor for PRRSVby monoclonal anti-idiotypic antibody, Mab2-5G2, which mimicks GP5antigen of PRRSV.The cell cycle is a cell proliferates from the end of last mitosis to the next complete mitosis,which refers to cell proliferation cycle. A standard cell cycle can usually be divided into fourcontinuous phases, Gl, S, G2and M phase. In the same kind of cells, different cells at differenttimes may be in the different cell cycle. In different cell cycles, the morphological andbiochemical characteristics the cell are different and the sensitivity to drugs, radiation andvirus infection is various. The objectives of this study were to syncronyze Marc-145cells todifferenct cell cyles and to analyze the levels of virus receptors and the infection of these ceilltypes with PRRSV.1. Development of a method for synchronization of Marc-145cells. By serumstarvation and drug blocking, we developed a method to synchronize Marc-145cells.Marc-145cells were synchronized to G0/G1phase when they were cultured in DMEMcontaining1%FBS for72h. After reseeding the synchronized cells in DMEM containing 10%FBS for17h, the cells were progressed into S phase. When cellsat S phase were treatedwith0.05μg/mL of Nocodazole for8h, the cells were synchronized to G2/M phase.2. Examination of PRRSV infection of Marc-145cell at different cell cycles. Marc-145cells which have been synchronizedto different cell cycles were inculated with PRRSV for thedetection of virus attachment and infection. The result showed that Marc-145cellsat G2/Mphase were favor to PRRSV infection than that at S phase.3. Expression levels of PRRSV receptors in Marc-145cellsat different cell cycles.Synchronized Marc-145cell at different cell cycle were harvested and expression levels ofdifferent PRRSV cellular receptorswere measured.The result showed that Marc-145cells atG0/G1phase had the highest expression level of CD163than that at other cell phases, andcells at G2/M phase had the highest expression level of NMHC-ⅡA than that at other cellphase. Marc-145cells at all phases had the same levels of vimentin.In Summary, these study suggest that PRRSV susceptibility to different cell cycle isdifferent, it maybe caused by the different PRRSV receptor expression level, this will be afoundation for the molecular mechanism of PRRSV infection research.