Mechanism Study Of Inhibition Of Stress Granules By Alpha And Gamma Coronaviruses

Infectious bronchitis virus(IBV)from ? coronaviruses is the first identified coronavirus in 1937 and is the major pathogen in poultry industry.?coronavirus Porcine Epidemic Diarrhea Virus(PEDV)has a nearly 100%mortality rate in suckling piglets,and Transmissible Gastroenteritis Virus(TGEV)infects piglets with severe diarrhea.They are still prevalent in poultry farms and swine farms,causing immense economic loses.Because coronaviruses are prone to mutate and recombine,existing vaccines still cannot prevent the long-term epidemics.The outbreak and pandemic of novel coronavirus from 2019 to 2020 indicate that coronaviruses not only harm animal breeding,but also pose a huge threat to public health.In depth research and recognition of coronaviruses are the basis for prevention and control of these epidemics.In this study,?and?coronavirus were used to study the host stress and interferon responses after infection.During RNA virus replication,viral double-stranded RNA(dsRNA)is detected by protein kinase PKR,in turn,PKR undergoes an auto-phosphorylation and phosphorylates the alpha subunit of eukaryotic translation initiation factor 2(eIF2).Phospho-eIF2?prevents the recycling of ternary complex tRNA^Met-Met-GTP-eIF2,and inhibits 43S translation complex formation,leading to translation shut off.The translation inhibition leads to polysome disassembly and the subsequent accumulation of the mRNAs associated with stalled ribosome complexes in cytoplasmic structures called stress granules(SGs).It is proposed that SGs promote interferon response.Some viruses have evolved various mechanisms to circumvent the formation of SGs,accomplishing efficient replication.However,it is unclear whether?and?coronaviruses harbor mechanism to antagonize the formation of SG.In this study,we examined the formation of SG by infection of IBV and PEDV and found there were only 20%infected cells with SGs,while 80%infected cells are absent of SGs.Besides,IBV and PEDV also impeded the formation of eIF2?dependent and independent SG triggered by different stress stimuli(heat shock,sodium arsenite and Na Cl).Further studies showed that IBV nsp15 inhibited the formation of SGs stimulated by various stress stimuli,and the endonuclease activity played a pivotal role.Moreover,the nsp15 from PEDV and TGEV also suppressed the formation of SG.Thus,the antagonism function of nsp15 to SG formation is universal phenomenon among different genus of coronavirus.Further study showed that the overexpressing nsp15 entered nucleus and retained the poly(A)binding protein(PABP1)in the nucleus,which is usually accompanied with mRNA nuclear retention.The ribonuclease activity of nsp15 is essential for nsp15 to suppress PABP1 nuclear export.Considering the RNA binding and cleavage capacity,nps15 probably targets to host nuclear mRNA,preventing host mRNA processing or nuclear export.We next constructed the nsp15 ribonuclease deficient recombinant IBV by mutating the enzymatic core residue histidine 238 to alanine.Compared with the wild type IBV,the nsp15 deficient rIBV-nsp15-H238A led to more dsRNA accumulation,strongly stimulated the phosphorylation of PKR and eIF2?,and effectively triggered the formation of SG,in concomitant with the higher production of IFN-?and viral replication reduction.The TBK1-IRF3-IFN signaling was attenuated in SG core protein G3BP1/2 knockout cells by rIBV-nsp15-H238A infection or poly(I:C)stimulation,demonstrating that SGs play an anti-viral role by promoting IFN signaling.In all,this study first reveals the novel biological role of coronaviruses nsp15 by suppressing the anti-viral SG,probably via targeting host mRNAs or cleaving viral dsRNA intermediates.