Inhibition Of Caprine Enterovirus Replication By Heat Shock Protein GRP78 Through Activation Of NF-κB Signaling Pathway

The interaction between hosts and viruses has always been a highly valued research topic in the field of virology.In-depth exploration of this relationship not only contributes to a better understanding of the functions of the host immune system,including processes such as virus recognition,activation of immune cells,and inflammatory responses but also aids in the development of more effective disease control and prevention strategies.These may involve studying virus transmission pathways,developing vaccines,and antiviral drugs,among other approaches.Caprine Enteroviruses(CEV),belonging to the Picornaviridae family,are members of the Enterovirus genus.Clinical manifestations of diseases caused by EV typically feature symptoms related to the respiratory,digestive,and nervous systems.Infections caused by caprine enteroviruses have emerged as newly reported infectious diseases in domestic livestock,leading to significant economic losses in caprine farming.While there have been reports on diagnostic methods,epidemiological backgrounds,and animal models related to caprine enterovirus infections,research on the pathogenic mechanisms and the interaction between the virus and host is still lacking.Heat shock protein GRP78,located in the endoplasmic reticulum,serves as a molecular chaperone.Apart from assisting in protein refolding and preventing protein aggregation to maintain cellular homeostasis,GRP78 also plays a crucial role in regulating cell vitality,proliferation,apoptosis,adhesion,as well as both innate and adaptive immune responses.Research has indicated that GRP78 can also function as a viral receptor,participating in infections by viruses such as Borna disease virus and Zika virus.However,there are few reports on whether this protein affects the replication of enteroviruses and its underlying mechanisms.To determine the impact of GRP78 on caprine enterovirus replication,this study utilized molecular biology techniques such as Western Blot and q PCR to measure the expression levels of GRP78 in virus-infected cells.The results demonstrated significant expression of GRP78 in enterovirus-infected cells.Through the construction of GRP78 overexpressing or knocked-down cells and employing methods such as dual-luciferase reporter genes,Western Blot,Co-IP,and laser confocal microscopy,the study investigated the influence of GRP78 on caprine enterovirus replication and its mechanisms.The findings revealed that GRP78 expression significantly regulates caprine enterovirus replication by recognizing the enterovirus 3D protein,binding with CHUK/IKBKB,and subsequently activating the NF-κB signaling pathway to inhibit caprine enterovirus replication.In summary,the study elucidates the regulatory role of GRP78 in caprine enterovirus replication and its mechanisms,providing new strategies for targeted drug therapy against caprine enterovirus infections.The specific research content encompasses three main aspects.1.Regulation of GRP78 Expression in Caprine Enterovirus Infection.Using methods such as q PCR and Western Blot,the expression of the heat shock protein GRP78 in Vero cells infected with caprine enterovirus was examined.Simultaneously,the duration of virus infection,different virus infection doses,and the pattern of GRP78 expression after virus infection were determined.The results indicate that in Vero cells infected with the virus,the m RNA level of GRP78 starts to increase at 4 hours post-infection,gradually rising with the duration of virus infection and reaching its peak at 12 hours.Similarly,the protein level of GRP78 begins to increase at 4 hours post-infection,gradually rising with the duration of virus infection and reaching its peak at 24 hours.Additionally,the expression of GRP78 in MDBK cells infected with enterovirus was examined,revealing a significant increase in both m RNA and protein levels after enterovirus infection.These findings suggest that enterovirus infection can stimulate the gene expression of host cell GRP78,with its expression positively correlated with the duration and dosage of virus infection.Importantly,this process does not exhibit cell dependency,laying the foundation for subsequent investigations into the influence of GRP78 on enterovirus replication.2.The Impact of GRP78 on Caprine Enterovirus Replication.Using gene overexpression and lentivirus-mediated gene knockdown techniques,cell lines with either overexpressed or knocked-down GRP78 were established.The success of GRP78 overexpression or knockdown was confirmed in 293 T cells through Western Blot and q PCR methods.Additionally,the viability of GRP78 knockdown cells was assessed using a CCK-F cell viability assay kit,revealing that the knockdown of GRP78 did not significantly affect cell viability.Various techniques including Western Blot,q PCR,and virus titration were employed to investigate the impact of GRP78 on enterovirus replication by examining the expression levels of the VP1 protein and virus titers in cells with GRP78 overexpression or knockdown after viral infection.The Western Blot results indicated an elevated expression of the viral VP1 protein in cells overexpressing GRP78,while q PCR and virus titration results showed a significant decrease in the transcription level of VP1 and virus titers compared to the control group.Conversely,in cells with GRP78 knockdown,Western Blot and q PCR results revealed an increase in the transcription and expression levels of the viral VP1 gene,and virus titration results indicated an elevated virus titer,indicating a clear influence of GRP78 expression on enterovirus replication.Further exploration of the interaction between GRP78 and enterovirus-encoded proteins was conducted using immunoprecipitation and laser confocal microscopy.The results showed that enterovirus 3D protein interacted with GRP78 and co-localized within the cells.Additionally,Western Blot and q PCR detection revealed that 3D protein could promote the expression of GRP78 protein.In contrast,enterovirus VP3,2A,and3 C proteins did not interact with GRP78 and could not enhance the expression of GRP78 protein in the cells.These findings suggest that GRP78 interacts with the enterovirus 3D protein,thereby inhibiting enterovirus replication,laying the foundation for a deeper understanding of enterovirus replication and pathogenic mechanisms.3.Mechanistic of GRP78’s Impact on Caprine Enterovirus Replication:Research has reported that GRP78 has a regulatory role in the NF-κB signaling pathway.To elucidate the relationship between enterovirus infection and GRP78,as well as the NF-κB signaling pathway,dual-luciferase gene reporter assays,laser confocal microscopy,and q PCR were employed.These methods were utilized to examine the impact of enterovirus infection on the NF-κB signaling pathway.The dual-luciferase gene reporter assay results indicated that enterovirus infection led to an increase in luciferase activity,which progressively rose with the duration and dosage of viral infection.This suggests that enterovirus infection may activate the NF-κB signaling pathway.Further laser confocal microscopy experiments revealed the nuclear translocation of p65 induced by enterovirus infection,confirming the activation of the NF-κB signaling pathway.q PCR results showed elevated expression levels of inflammatory factors downstream of the NF-κB signaling pathway,such as IL-6,IL-8,IL-1β,and TNF-α,after enterovirus infection,providing additional evidence of NF-κB pathway activation.These results collectively indicate that enterovirus infection can activate the NF-κB signaling pathway.Subsequently,dual-luciferase gene reporter assays,Western Blot,q PCR,and laser confocal microscopy were employed to investigate the impact of overexpressing enterovirus structural proteins(VP1,VP2,VP3,and VP4)and non-structural proteins(2A,2B,2C,3A,3C,and 3D)on the NF-κB signaling pathway.The results revealed that enterovirus 3D protein increased luciferase activity and induced nuclear translocation of p65,along with elevated expression levels of inflammatory factors such as IL-6,IL-8,IL-1β,and TNF-α.This indicates that enterovirus 3D protein can activate the NF-κB signaling pathway,and its ability to activate the NF-κB signaling pathway increases with higher transfection doses.In conclusion,enterovirus infection activates the host NF-κB signaling pathway,primarily through the activation exerted by the 3D protein.Simultaneously,to explore the relationship between GRP78 and the NF-κB signaling pathway,various techniques including Western Blot,q PCR,co-immunoprecipitation(Co-IP),and laser confocal microscopy were employed to assess the activation status of the NF-κB signaling pathway in cells with GRP78 overexpression and knockdown.The results indicated elevated levels of phosphorylated p65,significant degradation of IκBα,and increased expression levels of inflammatory factors IL-6,IL-8,and TNF-α in cells with GRP78 overexpression.This suggests that GRP78 may activate the NF-κB signaling pathway.Furthermore,treatment of GRP78 overexpressing cells with the NF-κB inhibitor Bay 11-7082 revealed that the inhibitor could suppress the increased phosphorylation of p65 and enhanced degradation of IκBα caused by GRP78 overexpression,providing additional evidence of GRP78 activating the NF-κB signaling pathway.Similarly,in cells with GRP78 knockdown,a decrease in the phosphorylation level of p65,attenuation of IκBα degradation,and reduced expression levels of inflammatory factors IL-6,IL-8,and TNF-α were observed.These results indicate that GRP78 can activate the NF-κB signaling pathway.Further experiments using immunoprecipitation and laser confocal microscopy confirmed the interaction and co-localization of GRP78 with the CHUK/IKBKB proteins in the NF-κB signaling pathway.These findings suggest that GRP78 activates the NF-κB signaling pathway through its interaction with CHUK/IKBKB.To determine the impact of GRP78 interacting with CHUK/IKBKB and activating the NF-κB signaling pathway on caprine enterovirus replication,Western Blot and q PCR methods were utilized to assess the expression levels of the virus VP1 protein and NF-κB signaling pathway-related proteins in cells with either GRP78 overexpression or knockdown upon enterovirus infection.The results revealed that in cells overexpressing GRP78 and infected with enterovirus,the expression level of the virus VP1 protein decreased,phosphorylation of p65 increased,degradation of IKBαintensified,and m RNA levels of IL-6,IL-8,and TNF-α elevated.Conversely,in cells with GRP78 knockdown and infected with enterovirus,the expression level of the virus VP1 protein increased,phosphorylation of p65 decreased,degradation of IKBαattenuated,and m RNA levels of IL-6,IL-8,and TNF-α decreased.These findings indicate that GRP78 can inhibit virus replication by activating the NF-κB signaling pathway.Furthermore,treatment of enterovirus-infected cells with GRP78 overexpression using the NF-κB inhibitor Bay 11-7082 showed significantly lower expression levels of the virus VP1 protein compared to the control group treated with Bay 11-7082,accompanied by a decrease in virus titers.Conversely,the expression levels of the virus VP1 protein in GRP78 overexpressing cells treated with Bay11-7082 were higher than the control group treated with DMSO,accompanied by an increase in virus titers.These results demonstrate that GRP78,through its interaction with CHUK/IKBKB proteins,activates the NF-κB signaling pathway,thereby inhibiting enterovirus replication.In summary,host cells respond to enterovirus infection by upregulating the expression of GRP78 protein.GRP78,by recognizing the enterovirus 3D protein and interacting with CHUK/IKBKB proteins,activates the NF-κB signaling pathway,ultimately inhibiting enterovirus replication.The results of this study reveal a novel mechanism of host heat shock protein GRP78 in regulating enterovirus and lay the foundation for further research on the interaction between host and virus,as well as the development of targeted drugs for enterovirus,providing theoretical support.