Identification Of HCMV IE86 Protein Complex By Coimmunoprecipitation And LC-MS/MS Analysis

Objective:1.To detect the expression of IE72 and IE86 in human embryonic lung fibroblasts cell MRC-5 and glioma cell line U87 MG cells infected with HCMV AD169.2.To screen proteins interacting with the IE86 protein by the method of co-immunoprecipitation coupled with LC-MS/MS analysis in MRC-5 and U87 MG cells.Methods:1.The MRC-5 and U87 MG cells were infected with HCMV AD169(MOI=1),and the changes of cell morphology were observed by inverted microscope.2.The MRC-5 and U87 MG cells were infected with HCMV AD169(MOI=1),then the culture supernatants were collected for virus titer determination at 6,12,24,48,72 h post-infection.The titer of virus was detected by the plaque assay.3.The MRC-5 and U87 MG cells were infected with HCMV AD169(MOI=1),and the total RNA and proteins were extracted.RT-PCR and Western Blot were used to test the expression of IE72,IE86 gene and proteins.4.The IE86 protein could be expressed in the HCMV-infected cells,the IE86 complex was purified from the infected cells via the method of co-immunoprecipitation with anti-IE86 specific antibody.Then SDS-PAGE and LC-MS/MS analysis werecarriedoutto separated and identified the components of the complex.To screen splicing factor interacting with IE86,the function of each protein was obtained from the specific database.Results:1.MRC-5 had obvious changes 6h post infection at MOI=0.1.The infected cells rounded and the intercellular space was wider.With the prolongation of infection time,the proportion of cells that had this morphology was increasing.Compared with MRC-5 cells,U87 MG did not show typical changes.At 24 h after infection,a small number of the cells were getting round.Results showed that permissive cells MRC-5show the cytopathic effect earlier than U87 MG.2.The titer of virus was detected by the plaque assay.MRC-5 and U87 MG cells were able to form an infectious virus.And,U87 MG replicated similarly to MRC-5.However,there was a difference in speed and quantity of virus replication.Obviously,completely permissive cells MRC-5 produced much more infectious virus than U87 MG.3.RT-PCR and Western Blot results show that the IE72 and IE86 genes and proteins expressed in MRC-5 were obviously higher compared with the corresponding hours in the U87MG(P<0.05),and the ratio of IE86/IE72 was higher in MRC-5 than U87MG(P<0.05).4.25 splicing factors interacted with IE86 were obtained from the study via co-immunoprecipitation and LC-MS/MS analysis in MRC-5 cells,and 8 splicing factors interacted with IE86 were obtained in U87 MG cells.Conclusions:1.The level of HCMV replication and the expression of IE72,IE86 is higher in MRC-5than U87 MG cells.2.In MRC-5 cells,25 splicing factors could be interacted with IE86,including hn RNPA2B1,hn RNPA1,hn RNPM,hn RNPK,hn RNPH1,hn RNPC and hn RNPA0 etc.;in U87 MG cells,8 splicing factors could be interacted with IE86,including hn RNPA2B1 and hn RNPA1,hn RNPM,hn RNPK,hn RNPH1,hn RNPC etc..3.Splicing factors may affect the splicing of IE m RNA,resulting in the difference in expression of IE in MRC-5 and U87 MG cells.