Studies On The Interaction Between Of HCMV-coding Protein PUL23 And Human Protein IFP35

Object:Human cytomegalovirus (HCMV) UL23 gene encoding virus tegument protein pUL23 is a member of virus US22 gene family.Currently pUL23 function is unknown.My studied group screened interaction of host proteins with the human cytomegalovirus protein pUL23 by yeast two-hybrid experiment. One interactional protein is IFP35 which is a interferon inducible protein. Virus infection causes the host's immune response, including the interferon response. It may be associated with the host immune response provoked by virus that interaction of viral protein pUL23 and interferon inducible protein IFP35. To determine whether the viral protein pUL23 relate to cellular interferon pathway, we use techniques of GST pull-down, immunoprecipitation and co-localization to further confirm protein interaction between pUL23 and IFP35, identificate interacting protein domain and the biological effects of interaction between pUL23 and IFP35. These results could provide the basis to study functions of pUL23.Methods:1) Determine the interaction between pUL23 and IFP35:â‘ Yeast two-hybrid experiment: The full length of UL23 and IFP35 were respectively constructed in the pGBKT7 and pGADT7 vectors and then used yeast two-hybrid system to determine the interaction of these two proteins.â‘¡GST pull-down experiment:Constructed the pGEX-4T-1-IFP35 vector, transformed it into E. coli strain Tuner (DE3) placI to expressed and purified GST-IFP35 protein. The same time, transfected pCDNA3.1(-)-HA-UL23 vector into Cos-7 cells and expressed HA-UL23 fusion protein, after hatching these two proteins, Westen blotting with HA antibody were excuted.â‘¢Immunoprecipitation experiment (Co-IP):Construction of pcDNA3.1(+)-IFP35-Flag vector, the two expression vector pcDNA3.1(+)-IFP35-Flag and pcDNA3.1(-)-HA-UL23 co transfected into Cos-7 cell, Westen blotting with HA and Flag antibody were excuted.â‘£Cells experiment of co-localization:Construction of pEGFP-Nl-UL23 and pDsRed2-C1-IFP35, these two expression vector were transfected into Hela cells, protein localization in cells by observated laser confocal microscope.2) Interaction domain of determining pUL23 and IFP35:27 different short pieces of UL23 were respectively constructed in the pGBKT7 and pGADT7 vector and then used yeast two-hybrid system to determine interactional region of pUL23, then the seven different IFP35 short pieces were respectively constructed in the pGBKT7 and pGADT7 vector, in order to confirme ineractional region of IFP35.3) Indentification of macromolecular complex by native polyacrylamide gel electrophoresis: pEGFP-N1-IFP35, pCMV-Nmi and pcDNA-HA-UL23 were transfected into Hela cells, supernatant fractions from cells were prepared and analyzed by Western blotting after running 6% native polyacrylamide gels.4) Westen blotting detect association among molecules:pcDNA-UL23-HA, pcDNA3.1(+), pcDNA-IFP35-Flag and pEGFP-N1 jointly transfected into HEK293T cells according to a certain percentage of plasmid dosage, Western blotting to test results.Result:1) Four methods of yeast two-hybrid system, Co-IP, GST pull-down and co-localization confirmed pUL23 can interact with IFP35.2) Co-localization of IFP35 and viral protein pUL23 is in the cytoplasm and surrounds nuclear observed by confocal microscopy.3) Important interactional regions of pUL23 are 594-624bp,while IFP35 are 80-268aa, that is NID domain.4) Nmi, IFP35 and pUL23 in Hela cells can form high polymer whoes size is about 300-400kDa.5) In HEK293 cells, with increase of the pUL23 expression, IFP35 expression was little increased.The expression of IFP35 influence the expression of pUL23. With increase of the IFP35 expression, pUL23 expression was obviously increased.Conclusion:My study determine the interaction between HCMV pUL23 and the host protein IFP35 by techniques of yeast two-hybrid system,GST pull-down, immunoprecipitation and co-localization. Experimental data show:Co-localization of IFP35 and viral protein pUL23 is in the cytoplasm and surrounds nuclear. pUL23 interact with the NID domain of IFP35. In the cells, pUL23, IFP35 and Nmi,which is another key interferon regulatory protein, form macromolecular. The expression of pUL23 in cells has relation with the expression of IFP35.The above mention results reveal the viral proteins pUL23 closely related to molecules involve interferon pathway in the host cell.It provided strong evidence to further study the relationship of viral proteins pUL23 and the immune system of host cells.