Study Of Molecular Mechanism Of SARS-CoV-2 And EV-D68 Encoded Protease Differentially Regulate Pyroptosis Of Host Cells

The epidemic of Corona Virus Disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-Cov-2)is a public health event with the widest spread and the most difficult to prevent and control in recent years.SARS-CoV-2 is an enveloped,single-stranded,positive-sense RNA(+ssRNA)virus,which belongs to the genus Coronavirus,which belongs to the family Coronaviridae and the order Nidovirales.SARS-CoV-2 can be transmitted by droplet,aerosol and contact.The main clinical symptoms of SARS-CoV-2 infection are fever,cough,respiratory distress,and in severe cases,even septicemia and organ failure.Pyroptosis plays an important role in the pathogenesis of SARS-CoV-2,and understanding the function of SRAS-CoV-2 encoded proteases in the pathogenesis could help to develop new therapeutic strategies.Enterovirus D68(EV-D68)is a nonenveloped,single-stranded,positive-sense RNA(+ssRNA)virus,which belongs to the Enterovirus genus of the Picornaviridae family.The biological features of EV-D68 are similar to rhinovirus but different from traditional enterovirus.EV-D68 are acid sensitive,and preference for lower growth temperatures.Compared to other enterovirus,which multiply in the intestine,EV-D68 multiply in the respiratory tract,and the modes of transmission primarily through the respiratory rather than fecal-oral route.There were only sporadic cases of EV-D68 since its identification and regular outbreaks occurred after 2014.EV-D68 infection can cause respiratory disease and is associated with a variety of central nervous system complications.To date,there is still a lack of safe and effective drugs or vaccines to control EV-D68 pandemic.Both SARS-CoV-2 and EV-D68 infection could induce and manipulate pyroptosis of host cells.Pyroptosis is an important way for host cells to resist viral infection and plays a critical role in the innate immune response.Upon activation by inflammatory stimuli or microbial infection,host gasdermins(GSDMs)undergo proteolytic cleavage by inflammasome-activated Caspases,leading to the formation of membrane pores results in pyroptotic cell death,which is characterized by the release of inflammatory cytokines and damage-associated molecular patterns(DAMPs)that promote an immune response against pathogens and recruit immune cells to the site of infection.The involvement of GSDM-mediated pyroptosis in the pathogenesis of various infectious and inflammatory diseases has been established.Accumulating studies suggest pyroptosis plays a significant role in the pathogenesis of viral diseases,including SARS-CoV-2,as well as other viral infections such as influenza,hepatitis,enterovirus and HIV.This study aimed to investigate the influence of viral-encoded proteins on pyroptotic activity during SARS-CoV-2 and EV-D68 infections,and to develop potential antiviral therapies against SARS-CoV-2 and other viral infections.Systematic research on the regulation of SARS-CoV-2 and EV-D68-encoded proteases on pyroptosis revealed the following:1.NSP5 protease alters the cellular localization of GSDMD and affects its function.We constructed an expression vector that fused GSDMD with red fluorescent protein,enabling real-time observation of the intracellular localization of GSDMD,and screening out that NSP5 protease changed the cellular localization of GSDMD.Immunoblotting assay results revealed that NSP5 cleaved GSDMD at residues Q29 and Q193 by its proteolytic activity.The cleavage of GSDMD by NSP5 disrupt the oligomerization of GSDMD-N terminal mediated by Caspase-1,thus inhibiting its function of pyroptosis.2.The cleavage of GSDME by NSP3 initiates GSDME-mediated pyroptosis.We screened out that NSP3 cleaved GSDME at residue G370 via its proteolytic activity,and initiates GSDME-mediated pyroptosis.Although sole expression of the NSP3-generated GSDME fragment(GSDME1-370)was insufficient to trigger pyroptosis,suggesting an additional role for NSP3 in initiating pyroptosis.3.3C protease cleaved GSDMDWe screened out that 3C protease encoded by EV-D68 cleaved GSDMD at residue Q193 via its proteolytic activity by immunoblotting assay,which reveal the different hydrolysis characteristics from the NSP5 protease encoded by SRAS-CoV-2.4.The cleavage of GSDME by 2A initiates GSDME-mediated pyroptosis.In screening for the effect of viral proteins encoded by EV-D68 on pyroptosis,we observed that 2A protease induced pyroptosis in He La cells significantly.Immunoblotting assay results revealed that 2A protease cleaved GSDME,indicating that 2A protease activated GSDME-dependent pyroptosis in host cells.We confirmed these results in the stable He La cell lines with down-regulated endogenous expression of GSDMD or GSDME.5.EV-D68 exhibited oncolytic effects via activating GSDME-mediated pyroptosis.Infection with EV-D68 caused pyroptosis on various types of cancer cells.Human lung carcinoma cells(A549 and Calu-3 cells)as well as rhabdomyosarcoma RD cells were infected with EV-D68,the results indicate significant pyroptosis induced by EVD68 infection across all tested cell lines.LDH release and GSDME cleavage were also observed in EV-D68 infection.The above results were validated in the stable Calu-3 cell lines with knockdown of GSDMD or GSDME.