Screening For Intraviral Protein-protein Interactions Of PEDV And Host Proteins That Can Interact With Nsp14、nsp16 By Yeast Two-hybrid System

Porcine epidemic diarrhea(PED) is an acute and highly contagious intestinal disease caused by porcine epidemic diarrhea virus(PEDV). The main characteristics of the disease are acute enteritis,vomiting,diarrhea and dehydration. Pigs of all ages are susceptible and newborn piglets are most susceptible, high morbidity and mortality. PED has caused great economic loses in the pig industry.Yeast two-hybird system is an effective technique to study protein interactions. The system is highly sensitive and could detect weak protein interactions.In this study, we explore the intraviral protein-protein interactions of PEDV and host proteins that can interact with nsp14、nsp16 by yeast two-hybrid system. The main research contents are as follow:1.The analysis of intraviral protein-protein interactions of PEDV by Yeast two-hybirdFirst of all, we constructed two sets of Yeast recombinant plasmids of all viral proteins of PEDV except for nsp11, including p GBKT7-X(bait) and p GADT7-X(prey)(totally 44 recombinant plasmids). By autoactivation and toxicity testing, we found recombinant plasmid p GBKT7-nsp15、p GADT7-nsp4、p GADT7-nsp5、p GADT7-nsp15 had toxicity to Y2 H and p GADT7-nsp3-1 had autoactivation(cannot be used for Yeast two-hybrid). Then, we transformed all bait and prey into Y2 H in a point-to-point way to screen for interviral protein-protein interactions. In total, 462 pairs of interactions were screened by Yeast two-hybird system. As a result, 46 pairs of interactions were detected. 29 of all were in one direction. 12 of all were in both directions and 5 of all were self-interaction.2. Construction and identification of Yeast two-hybrid c DNA library of porcine intestinal epithelial cellsPorcine intestinal epithelial cells were grown in vitro and we extracted the total RNA. Then we used the total RNA as template to created a ds-c DNA pool with end sequences homologous to p GADT7-Rec. We transformed the ds-c DNA pool and p GADT7-Rec into Y187 Yeast Strain and allowed the yeast to perform a recombination step between the linear prey vector p GADT7-Rec and the ds-c DNA to get the c DNA library. Colonies were pooled, mixed, and aliquoted into multiple vials. Each single 1ml vial can be used for a two-hybrid screen. In this study, we constructed and identified a high-quality Yeast two-hybrid c DNA library of porcine intestinal epithelial cells. The titerof library is 6.5×108 cfu/m L, and the capacity is 1.04×1011cfu.3.Screening the c DNA library for host proteins that can interact with nsp14、 nsp16We transformed p GBKT7-nsp14 and p GBKT7-nsp16 into Y2 H strain respectively to prepare a concentrated overnight culture of the bait strain. Then we combined the c DNA library with the two bait Strains respectively to screen for host proteins that could interact with bait. The results in yeast system showed that host proteins CCT7, EIF3 I interact with nsp14 and host proteins DDIT4, UBE2 K, UBA52 interact with nsp16. In order to further verify whether the interactions could happen in mammalian cells, we performed Co-immunprecipitation experiments in HEK293 T cells. The results showed that host proteins DDIT4, UBE2 K interact with nsp16 and there is no direct interactions between nsp14 and host proteins. Further we detected co-localization between nsp16 and DDIT4 in cytoplasm by indirect immunofluorescence, and we also detected co-localization between nsp16 and UBE2 K in cytoplasm.