African Swine Fever Virus MGF110-7L Induces Host Cell Translation Suppression And Stress Granule Formation By Activating The PERK/PKR-eIF2α Pathway

African swine fever(ASF)is the "number one killer" in the global pig industry.Until now,there has been no effective vaccine and medicine,which can only be controlled by strict quarantine and culling measures.The limited understanding of the biological characteristics of African swine fever virus(ASFV)is the main reason that the research and development of vaccines and drugs for African swine fever has been hindered.The virus is completely dependent on the host translation system to synthesize viral proteins,so the strategy of "hijacking" the host translation system is the key to determining viral infection.As a core node in the regulation of translation initiation,translation initiation factor eIF2α can control protein translation reprogramming and stress response direction,and has important effects on virulence,tropism and pathogenicity of viruses.However,there is a lack of understanding about the translation regulation strategies of ASFV.This study focuses on how ASFV interacts with the eukaryotic translation initiation Factor 2α(eIF2α)signaling pathway.The main contents are as follows:First,the ASFV encoding proteins that significantly induced eIF2α phosphorylation was screened.The reporter plasmids of ATF4-RLuc and ATF4-EGFP were constructed.The luciferase reporter gene detection and western blot technique were used to detect the early ASFV protein MGF110-7L,which significantly increased the phosphorylation level of eIF2α and up-regulated the expression of activated transcription factor ATF4,and activated the cell integration stress response.Secondly,the function of MGF110-7L was verified.Phosphorylation of eIF2α affects host cell translation,stress particle formation and cell cycle.In order to investigate the effects of MGF110-7L on host cell translation and stress particle formation,laser scanning confocal microscopy and western blotting were used to find that MGF110-7L induced host cell translation block and stress particle formation.Moreover,ISRIB treatment could restore the host translation block and stress particle formation induced by MGF110-7L,suggesting that MGF110-7L induced host cell translation block and stress particle formation through eIF2α phosphorylation.Furthermore,the mechanism of eIF2α phosphorylation induced by MGF110-7L was studied.Biochemical tests showed that overexpression of MGF110-7L protein activated protein kinase R(PKR)and PKR-like endoplasmic reticulum(ER)kinase(PERK),GSK2606414 and C16 treatment could restore MGF110-7L induced eIF2α phosphorylation.These results suggest that MGF110-7L induced eIF2α phosphorylation is mediated by PKR and PERK,which is important for host translation block and SGs formation.Sequence alignment analysis showed that the amino acid sequence of MGF110-7L protein from 12 highly pathogenic strains in different years and different regions was highly conserved,and the endoplasmic reticulum retention signal-KKDEF motif was present in the carboxyl terminal.Laser scanning confocal microscopy demonstrated that MGF110-7L protein was mainly localized in the endoplasmic reticulum and led to specific recombination of secretory pathways,suggesting that MGF110-7L may serve viral proliferation by regulating intracellular protein secretion pathways or related membranous organele components.Further proteomic analysis showed that MGF110-7L interacts with host proteins involved in regulation of endoplasmic reticulum homeostasis.Finally,MGF110-7L protein was found to induce endoplasmic reticulum stress.Using laser scanning confocal microscopy and western blotting,MGF110-7L was found to induce endoplasmic reticulum stress in host cells and activate the unfolded protein response IRE1-XBP1 branch.Cells induced eIF2α phosphorylation through kinase PERK and PKR to induce translational block and stress granules formation.In conclusion,the structure,localization and potential functional characteristics of MGF110-7L,a multigene family protein of ASFV,were studied for the first time in this paper,and the stress relationship between MGF110-7L and the protein translation system of host cells was revealed for further understanding...