The Mechanism Of MERS-CoV Nsp4 Inhibits The Activation Of NLRP3 Inflammasome

Research BackgroundMiddle East respiratory syndrome coronavirus(MERS-CoV)causes severe respiratory disease,which has high fatality rate about 35%,first found in the Middle East area in 2012.The clinical manifestations of MERS-CoV infection range from asymptomatic infection to mild,moderate and severe disease,often accompanied by severe pneumonia,acute respiratory distress syndrome(ARDS),septic shock and multiple organ failure.Cytokine storm phenomenon often appears in viral infection,a large amounts of pro-inflammatory cytokines such as IL-6,TNF?,IL-1? and other pro-inflammatory factors are produced in a short period of time,causing serious damage to the body tissues and organs,in which IL-1? is produced by the activation of inflammasome.NLRP3 inflammasome is cytosolic multiprotein complex,which plays an important role in regulating inflammation during virus infection.During viral infection,NLRP3 inflammasome is activated,which can trigger the secretion of pro-inflammatory cytokines IL-1? and IL-18,and induce inflammatory death known as pyroptosis.The activation of NLRP3 inflammasome requires two signals.The first signal is usually provided by microbial components or endogenous cytokines,binding to Toll-like receptors(TLRs)or cytokine receptors(IL-1R or TNFR)to promote the activation of NF-?B.The second signal can be provided by a variety of stimuli,causing K+efflux,Ca2+signaling,ROS production,mitochondrial damage and lysosomal rupture,that activate NLRP3 inflammasomes.MERS-CoV infection induces the activation of inflammasome,but the role of viral proteins in the activation and regulation of inflammasome remains unclear.In this study,macrophages stimulated by LPS and ATP were used as the NLRP3 inflammasome activation model.We aimed to find MERS-CoV non-structural proteins that influence inflammasome activation,and then investigate the underlying regulatory mechanism,providing theory on pathogenic mechanism of MERS-CoV and target for developing drugs.Aims1.Screen MERS-CoV nsp that can affect the activation of NLRP3 inflammasome.2.Explore the mechanism of nsp affecting the activation of NLRP3 inflammasome and its significance in virus infection.3.Provide new target for the prevention and treatment of MERS-CoV.Methods1.Construct the MERS-CoV non-structural protein nsp1-nsp 16 overexpression vectors,package the lentivirus by the calcium phosphate method,collect the viral supernatant,and infect THP-1 cells to obtain viral protein stably expressed cells.2.Detect the level of IL-1? secreted by macrophages by ELISA,detect the expression of cleaved caspase-1(p20)by Western Blot,and detect the level of cell pyroptosis by fluorescence microscope after PI staining.3.Detect the activation of NF-?B in THP-1 cells and the expression of NLRP3 inflammasome-related proteins by real-time fluorescent quantitative PCR and Western Blot.4.Detect the expression of LC3B protein by Western Blot,observe the formation of autophagosome with transmission electron microscope,and observe the formation of GFP-LC3 autophagosome fluorescent spots with laser confocal microscope.5.After blocking the autophagy process by HCQ,we used Western Blot to detect the expression of LC3B protein and the activation of caspase-1.6.Observe the localization of nsp4 in cells and the co-localization of nsp4 with NLRP3,ASC and pro-caspase-1 with laser confocal microscope.7.Detect the interaction between nsp4 and NLRP3 inflammasome proteins NLRP3 and ASC by Co-IP.8.Observe the effect of nsp4 on the formation of ASC spots with confocal microscope.9.Detect the effect of nsp4 on the interaction between NLRP3 and ASC by Co-IP;10.Detect the effect of nsp4 on VSV replication by real-time fluorescent quantitative PCR,observe the effect of nsp4 on VSV titer by fluorescence microscope,use CCK-8 to detect the effect of VSV on the proliferation of THP-1 cells expressing nsp4.Results1.Construction of MERS-CoV non-structural protein nspl-nsp16 overexpression lentiviral vectorsMERS-CoV non-structural protein nspl-nspl6 was constructed on PCDH vector,and the calcium phosphate transfection method was used for lentivirus packaging in 293T cells.The virus supernatant was collected and successfully infected THP-1 cells to stably express MERS-CoV non-structural protein.2.nsp4 inhibits the activation of NLRP3 inflammasomeThe macrophages were treated with LPS/ATP to construct the NLRP3 inflammasome activation model.Compared with the control group,nsp4 significantly inhibited the secretion of IL-1? and the activation of caspase-1.PI staining was used as a marker of cell pyroptosis,and fluorescence microscopy showed that nsp4 inhibited cell pyroptosis.3.nsp4 does not affect the activation of NF-?B(first signal)Compared with the control group,nsp4 did not affect the activation of NF-?B in macrophages treated with LPS.There was no significant difference in the protein level of p-p65,the mRNA levels of pro-IL-1? and NLRP3,suggesting that nsp4 inhibited the activation of NLRP3 inflammasome was not through affecting the activation of NF-?B.4.nsp4 causes autophagyCompared with the control group,nsp4 induced the increase of autophagy-related genes,and the transformation level of LC3B I to LC3B II was increased.The generation of bilayer autophagosomes was observed by transmission electron microscopy.After the autophagy process was blocked by HCQ,the conversion of LC3B I to LC3B II was decreased,and cleaved caspase-1 activation was not significantly changed,suggesting that nsp4-induced the inhibition of NLRP3 inflammasome was not caused by promoting autophagy.5.nsp4 interacts with NLRP3 inflammasome complexImmunoprecipitation showed that nsp4 could interact with the NLRP3 inflammasome complex(NLRP3,ASC and caspase-1).Laser confocal microscopy showed that nsp4 was distributed in the cell membrane,nucleus and cytoplasm.6.nsp4 inhibits the assembly of NLRP3 inflammasomePerinuclear ASC spots were observed in LPS/ATP-stimulated macrophages,which are marker of inflammasome activation.Compared with the control group,the spots produced by nsp4 group were smaller and scattered,indicating that nsp4 inhibited the formation of ASC spots.7.nsp4 inhibits NLRP3 inflammasome activation and promotes VSV replication We selected the VSV virus that can induce NLRP3 inflamamsome activation to investigate the effect of nsp4 on virus replication.Q-PCR results showed that the mRNA level of VSV in nsp4 group was higher than that of the control group 24 h after VSV infection.CCK-8 results showed that VSV infection significantly inhibited cell proliferation in the nsp4 group compared with the control group.These results indicated that nsp4 promoted viral replication,which would result in the inhibition of cell proliferation after massive viral expansion.Conclusion and significance1.In this study,we found that MERS-CoV nsp4 could inhibit the activation of NLRP3 inflammasome and the secretion of IL-1? and cell pyroptosis.2.nsp4 induced autophagy,but autophagy is not the reason by which nsp4 inhibited the activation of NLRP3 inflammasome.3.nsp4 interfered with the assembly of inflammasome and affected the activation of inflammasome by interacting with the NLRP3 inflammasome complex.4.nsp4 inhibited NLRP3 inflammasome activation,benefiting viral replication.In this study,we found MERS-CoV nsp4 inhibited IL-1? secretion and pyroptosis by affecting the assembly of NLRP3 inflammasome.We provided a novel mechanism that MERS-CoV promoted its replication via interfering the inflammatory response in host cells.These findings indicated nsp4 is a potential target for anti-MERS-CoV,and regulating the activation of NLRP3 inflammasome might be an effective strategy for control MERS-CoV infection.