| Porcine respiratory and reproductive syndrome virus?PRRSV?is a major pathogen that causes severe reproductive disorders and respiratory system disease of pigs.The outbreak and spread of the disease causes the severe economic losses to pig industry worldwide.PRRSV is an enveloped single stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales.PRRSVs include two serotypes:Europe type?or PRRSV-1?and America type?or PRRSV-2?.In China,the major circulating type is America type and very few reports about Europe type.The main susceptible cell of PRRSV is porcine alveolar macrophages?PAM?,additionally,african green monkey kidney cells?MARC-145?is commonly used to separate and cultivate PRRSV.PRRSV infection in host cells is mediated by receptor-and clathrin-mediated endocytosis.CD163 is an important receptor that determines the cell tropism of PRRSV and helps viral uncoating.Non-muscle myosin heavy chain type II A?NMHC IIA or MYH9?is another indispensable cellular factor discovered by our laboratory.Sialoadhesion?or Sn,CD169?,Heparan sulphate?HS?,vimentin?Vim?,CD151,and DS-SIGN?CD209?play important roles in the process of virus attachment and internalization.MYH9 is a kind of cytoskeletal protein,involved in cell migration,cell mitosis,vesicle metastasis and secretion and goods transportation on microfilament bundle.MYH9 acts like cell receptor that was identified using Mab-5G2 by co-IP and mass spectrometry.According to the C terminal sequence,MYH9 was divided into three subtypes:type IIA,IIB and IIC.Previous results showed that the C terminal of MYH9?named PRA?specifically combined PRRSV GP5 protein and promoted virus internalization.Further studies found that MYH9 promotes PRRSV enter host cells acting as a co-factor with CD163.The objectives of this study are to determine interaction between MYH9 and PRRSV GP5 and the formation of the protein complex,and to explore MYH9's role in the early stage of PRRSV infection in host cells.The contents of this research are as follow:1.The study on PRRSV SD16 strain GP5 truncated proteins.Construction of prokaryotic expression plasmid:pET-28a-GP5?-His and then GP5?inclusion body was expressed in the E.coli.Purified protein was refolded by dialysis against buffer containing a gradient of urea concentrations.PAM cells and MARC-145 cells were incubated with prokaryotic expression of GP5?-His protein,and then infected with PRRSV SD16.We found that GP5?-His protein can block most of the virus.Using the same method,GP5-ecto-1-His and GP5-C-His proteins were purified and also did virus blocking experiment.The results show that only GP5-ecto-1 can block PRRSV and for other strains,including JXA1,VR2332,G073-3 and GZ11-G1,GP5-ecto-1 also suppress virus infection except CH-1a.By analyzing CH-1a GP5-ecto-1 sequence,we found that the F39 amino acid of CH-1a GP5 contain a benzene ring on side chain,while other viruses don't have.Therefore,GP5-ecto-1 function as blocking most of PRRSV infection and the GP5-ecto-1region is very important for virus to combine host cells.2.The study on GP5 and MYH9 interaction.The soluble GP5?and PRA kept in our lab were respectively bond in ELISA 96 wells or PVDF membrane as"bait protein".Next,PRA or GP5?were incubated in wells or PVDF membrane.5G2 or anti-His antibody were used to detect the combined protein.Compared with the negative control protein sp239,GP5?-PRA or PRA-GP5?have obvious interaction.In order to further study if GP5glycosylation and sialic acid modification influence GP5-PRA interaction.we use MARC-145-GP5Flag cell line,stably express GP5,which was constructed in our lab previously.Cell lysates were treated with PNGaseF or Neuraminidase to remove GP5glycosylation and sialic acid modification.Then we use anti-Flag antibody to combine GP5and also precipitated MYH9 from cells.Overall,GP5-MYH9 contain both linear epitope and conformational epitope and the interaction have nothing to do with GP5 glycosylation and sialic acid modification.To identify GP5 region that combine MYH9,co-IP and His pull down were used.The results show that GP5 and GP5-N can interact with MYH9?or PRA?not GP5-C.For far western experiment,GP5-ecto-1 was further proved directly combine PRA.GP5-ecto-1 induce MYH9 form aggregation in MARC-145 cells,and also,in vitro,prokaryotic expression GP5-ecto-1/PRA protein complex was observed form thick protein bundle while PRA or GP5-C/PRA didn't.When add S100A4 into GP5-ecto-1/PRA complex,MYH9 protein bundle was disassembled immediately.Additionally,co-IP and His pull down results show that S100A4 can competitively bind to MYH9 when S100A4 was added into GP5-ecto-1/MYH9?or PRA?complex.3.The study on MYH9's role during the process of PRRSV infection.MARC-145 and PAM cells was infected with 50 MOI SD16 strain with adsorption for 2 h and then turn to37?.Cells were observed at 0 min,5 min,15 min and 30 min respectively using laser confocal microscope.Co-localization of PRRSV virions with endogenous MYH9 and MYH9 filament assembly into aggregates were both observed by 15 min?in MARC-145cells,15min and 30min in PAM cells?after the temperature shift to 37°C.Notably,after the temperature shift,increased PRRSV N protein levels were detected at 15 min.These findings indicated that MYH9 aggregation is required for PRRSV internalization.Endogenous S100A4 in MARC-145 cells during PRRSV internalization was detected by qPCR,western blot and confocal.These results show no significant change in S100A4.When S100A4overexpressing in MARC-145,PRRSV SD16 strain infection was obviously inhibited at early stage?within 30min?.The effect was also found in other strains of PRRSV.These results indicate the S100A4 act as potentially antiviral drug.To sum up,this research mainly focuses on MYH9 interact with PRRSV GP5-ecto-1protein and its role in virus infection.These findings support the conclusion that MYH9filament aggregation is required for PRRSV internalization and subsequent viral infection.Above studies provide a new research direction and contents on PRRSV infection. |
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