Identification And Analysis Of Host Cell Proteins Interacting With Porcine Epidemic Diarrhea Virus NSP8

Porcine epidemic diarrhea(PED)is caused by porcine epidemic diarrhea virus(PEDV),an contagious disease,which takes economic losses to pig industry.PEDV is a single-stranded RNA virus containing seven open reading frames(ORF)in which ORF1 a and ORF1 b encode two polyproteins PP1 a and PP1 ab.These two proteins are cleaved into 16 non-structural proteins(NSPs).NSPs are active enzymes that play important roles in virus replication and transcription.This study analyzed the interactions between PEDV NSP8 and host cell proteins,the main research contents were as follows:(1)NSP8 is a highly conserved protein according to the result of full genetic analysis and sequences alignment and high-titer polyclonal antibody was prepared which could specifically recognized PEDV NSP8.(2)pGBKT7-nsp8 was used as a bait to hybridize with porcine kidney cells cDNA library.By two rounds of screening experiments with selective media and cross-validation assay,four host proteins: LDHB,SERTAD3,GMS2 and PSMB10 were proved to interact with NSP8.According to the researches,the above proteins are all involved in intracellular signaling pathways.In addition,LDHB plays an important role in the process of glucose metabolism and promotes autolysis of cells after acidification of lysosomes.(3)With Co-Immunoprecipitation and Indirect Immunofluorescence Assay,we figured out that these two proteins had interactions with each other.Meanwhile,NSP8 was located in nucleus and cytoplasm.Above all,the interactions between LDHB and NSP8 were screened and identified by yeast two-hybrid system,Co-Immunoprecipitation and Indirect Immunofluorescence Assay,which laid a foundation for understanding the interaction mechanism between PEDV and host cell proteins,and provided scientific data for elucidating the mechanism of PEDV infection.