The Interaction Mechanism Between Porcine Epidemic Diarrhea Virus And Host Stress Granules

Porcine epidemic diarrhea virus（PEDV）is anα-coronavirus causing severe diarrhea in suckling piglets and the mortality rates are up to 80%-100%,which endangers the development of pig husbandry.Like other coronaviruses,the genome of PEDV is susceptible to mutation and there is no effective vaccine or specific therapeutic drugs currently.Hence,it is particularly important to fully understand the interaction mechanism between host cells and PEDV infection and to develop antiviral targets from the perspective of the host.Since PEDV replication is completed in the cytoplasm and is completely dependent on the host translation system,it is a relatively scientific research strategy to search for antiviral targets from the perspective of the host translation system.Stress Granules（SGs）is a protective mechanism for environmental stimuli（including virus infection）produced when the translation system is regulated in eukaryotic cells.When it happened,protein translation was suspended,translation initiation complexes with 40S ribosomal subunit and many RNA binding proteins in the cytoplasm gathered together to form dense granules.It has been reported that PEDV infection can induce the formation of SGs in infected cells.However,the interaction mechanism between PEDV and SGs is unknown.Therefore,study the molecular mechanism of PEDV regulation of SGs is of great significance to the research and development of anti-PEDV drugs and prevention and control of PEDV.1.PEDV infection induces transiently SGs formation.Under normal circumstances,SGs components are dispersed in the cytoplasm.However,under stress conditions,these components aggregate to form SGs.In Immunofluorescence（IFA）,the former presents homogeneous staining,while the latter presents granular staining.G3BP1 is a key protein that triggers the assembly of the core structure of SGs,and is often used as a marker protein to detect SGs.In this study,distribution of G3BP1 was determined by IFA and found that the proportion of SGs positive Vero-E6 cells（infected by PEDV for≤18 h）of the total number of PEDV infected cells increased gradually,and then（infected by PEDV after18 h）decreased.Similarly,PEDV infected Hu H-7 cells showed similar results.In addition,cells infected with PEDV for 24 or 36 h were transfected with the SGs inducer Poly（I:C）,and IFA results showed that the formation of SGs mediated by Poly（I:C）could also be significantly inhibited in the later stage of PEDV infection.It indicates that PEDV infection induces the formation of SGs in cells in the early stage,but can eliminate SGs in the later stage.To analyze the influence of SGs on PEDV replication,PEDV infected Vero-E6 cells or Hu H-7 cells which overexpressed EGFP-G3BP1 protein.RT-PCR and Western-Blot results showed that overexpression of EGFP-G3BP1 could inhibit the m RNA transcription or protein expression level of PEDV-N;Virus plaque formation assay showed that overexpression of EGFP-G3BP1 could significantly reduce the virus titer.In contrast,when G3BP1 was silenced in Vero-E6 or Hu H-7 cells and then infected with PEDV,knockdown of G3BP1expression was shown to increase the m RNA transcription or protein expression level of PEDV-N and viral titer.The above results indicate that the SGs induced by PEDV are not benefit for PEDV replication,so the SGs is eliminated in the later stage of PEDV infection to ensure the PEDV replication.2.Caspase-8 activation mediated G3BP1 cleavage in the later stage of PEDV infection.Some viruses encode a protease that cleaves G3BP1 to disassemble SGs.To reveal the mechanism of inhibition of SGs in the later stage of PEDV infection,G3BP1 protein level was detected during PEDV infection.The C-terminal epitope antibody of G3BP1 was used for Western-Blot detection,and it was found that 68 k Da G3BP1 protein appeared in Vero-E6 cells within 36 h after PEDV SQ2014 isolated strain infection,but about 40 k Da bands also appeared at 24 and 36 h,named as G3BP1_cpc（C-terminal cleavage products）.In addition,the PEDV classical vaccine strain CV777 and isolates strain HLJBY also showed this small molecular weight band at 24 and 36 h after infection.These results indicate that G3BP1protein could be cleaved in the later stage of PEDV infection without virus strain specificity.Then,cells were transfected with eukaryotic plasmid Flag-G3BP1-HA（Flag-labeled N terminal and HA-labeled G3BP1 C terminal）and infected with SQ2014,Western-Blot assay was performed with Flag-labeled and HA-labeled G3BP1,respectively.Except for full-length G3BP1,Flag-labeled antibodies could recognize cleaved bands around 27 k Da,while HA antibodies could recognize G3BP1_cpc.Therefore,the PEDV induced G3BP1 clelavage sites are closer to the N-terminal.In later stage of PEDV infection,Caspase protease was activated to induce apoptosis.To check whether G3BP1 cleavage was related to apoptosis,Vero-E6 cells were treated with apoptosis inducers CPT and TSA.Western-Blot results showed that G3BP1_cpc also appeared in Vero-E6 cells treated with CPT and TSA.Importantly,pretreatment with the pan-caspase inhibitor Z-VAD-fmk inhibited the cleavage of G3BP1 in CPT treated or SQ2014 infected cells.Then,Western-Blot analysis results showed that cleaved Caspase-8 bands（p43 and p18）appeared at 18 h after SQ2014 infection,indicating that Caspase-8 was activated in the later stage of PEDV infection.Further study showed that Caspase-8 inhibitors inhibited endogenous G3BP1 cleavage in PEDV-infected cells,while overexpression of Caspase-8enhanced exogenous G3BP1 cleavage.Moreover,purified prokaryotic expressed Caspase-8and G3BP1.In vitro protease acitivity assay showed that Caspase-8 directly cleaves G3BP1protein.In summary,Caspase-8 is activated to mediate SGs core protein G3BP1 cleavage in the later stage of PEDV infection.3.Caspase-8 mediated G3BP1 cleavage inhibits SGs to promote PEDV replicationTo explore the influence of G3BP1 cleavage on SGs and its role in PEDV replication,the cleavage site of G3BP1 should be determined firstly.The potential cleavage sites of G3BP1 were predicted according to the molecular weight of the cleavage products of G3BP1,and the prokaryotic expression mutant of G3BP1 was constructed by site-directed amino acid mutagenesis.The purified G3BP1 mutants were detected with Caspase-8 for protease activity in vitro,and it was found that Caspase-8 cleaved G3BP1 by aspartic acid（D168 and D169）at positions 168 and 169.To verify whether the cleavage site of G3BP1 in cells with PEDV infection is consistent with that in vitro,eukaryotic G3BP1 single site mutants（EGFP-G3BP1-D168E and EGFP-G3BP1-D169E）and double sites mutant（EGFP-G3BP1-D168/169E）were constructed.Then,cells with G3BP1 wild-type and mutants overexpressed were infected with PEDV,and Western-Blot was performed with GFP antibody.The results showed that EGFP-G3BP1 protein of about 100 k Da and about 55 k Da bands were detected in both the overexpressed wild-type and single site mutant cells,while only about 100 k Da EGFP-G3BP1 protein was detected in the over-expressed double sites mutant cells.These results indicated that the G3BP1 double sites mutant was not cleaved in PEDV-infected cells.It has been reported that G3BP1 lost the ability to form SGs after cleavage.To detect whether the cleavage of G3BP1 at position 168 and 169 D affected the formation of SGs,G3BP1 mutants and G3BP1_cpn（the N-terminal truncated G3BP1）and G3BP1_cpc（the C-terminal truncated G3BP1）were overexpressed in cells,and the results showed that only the truncated G3BP1 could not form SGs.To study the influence of G3BP1 cleavage on SGs formation during PEDV infection,wild type,single site and double sites mutants of G3BP1were transfected into cells and then PEDV was infected.IFA results showed that only the level of SGs in cells with double sites mutant overexpressed were not inhibited.RT-PCR and Western-Blot results showed that overexpressed double sites mutant could inhibit the m RNA transcription or protein expression level of PEDV-N.The results of the virus plaque formation assay showed that overexpression of the double sites mutant could significantly reduce the virus titer.To detect the correlation between the activity of Caspase-8 and SGs and PEDV replication,cells were infected with PEDV after treatment with Caspase-8inhibitors,and the results showed that Caspase-8 inhibitors could maintain the level of SGs and inhibit the replication of PEDV in the later stage of PEDV infection.In summary,PEDV activated Caspase-8 mediated G3BP1 cleavage inhibits the ability of cells to form SGs in the late stage of infection to promote the proliferation of PEDV.