Screening and identification of host proteins interacting with Nsp8 of porcine epidemic diarrhea virus

Abstract:

Porcine epidemic diarrhea virus (PEDV) is an enterovirus that can cause severe diarrhea and dehydration.The widespread epidemic of PEDV has caused huge economic losses to the pig breeding industry,which,however,lacks effective means for prevention and treatment.Nsp8 is an important non-structural protein involved in the replication of PEDV,while the host proteins interacting with Nsp8 remains unclear.[Objective] To screen the host proteins interacting with PEDV Nsp8 and explore the effects of the host proteins on the replication of PEDV,so as to provide a theoretical basis for discovering new key functional receptors or therapeutic targets of PEDV.[Methods] The eukaryotic expression plasmid of PEDV Nsp8 was successfully constructed with the eukaryotic expression vector pcDNA3.1(+).The host proteins interacting with PEDV Nsp8 were screened by co-immunoprecipitation,mass spectrometry,and laser confocal microscopy.The effects of the host proteins on PEDV replication were explored by overexpression and knockdown in LLC-PK cells.[Results] Thirty-six potential host proteins interacting with Nsp8 were screened by mass spectrometry,and the interaction between heat shock protein member 8(HSPA8) and Nsp8 was verified.The overexpression of HSPA8 in LLC-PK cells inhibited the overexpression of Nsp8 in a dose-dependent manner.Meanwhile,it significantly inhibited the replication of PEDV in a dose-dependent manner at the protein and transcriptional levels.Interfering with endogenous HSPA8 expression significantly promoted the replication of PEDV.The 50% tissue culture infectious dose (TCID₅₀) and indirect immunofluorescence further proved that HSPA8 inhibited PEDV replication.[Conclusion] This study screened out the host protein HSPA8 interacting with PEDV Nsp8 and proved that HSPA8 could significantly inhibit PEDV replication,which provided a new idea for the design of HSPA8-targeted drugs for the prevention or treatment of PEDV.

Key words:porcine epidemic diarrhea virus;Nsp8;co-immunoprecipitation;HSPA8