Mfn2 Promotes SYVN1-dependent K48 Ubiquitination Of MAVS To Increase The Susceptibility Of Influenza Virus

The theory of"emotional illness"in Chinese medicine has increasingly shown important value and significance in the etiology of modern diseases.Emotional stress can cause the body's susceptibility to a variety of diseases by breaking the stability of the internal environment.In addition to the virus's own factors,whether the host is in a"susceptible"state also has an important impact on the development of influenza virus infection.Mitochondrial antiviral-signaling protein?MAVS?-mediated IFN?secretion is a key“susceptibility”factor affecting host RNA infection.Our previous study found that restraint stress load and stress hormone corticosterone?CORT?can reduce the protein expression level of MAVS in mouse lung tissue,reduce the secretion of IFN?,and thus increase the susceptibility of mice to influenza virus.And our preliminary studies suggest that the reduction in MAVS protein is associated with ubiquitination.In this study,the in vivo and in vitro influenza virus susceptibility model established by mouse restraint stress load and CORT load A549 cells was used to study the mechanism of stress-induced ubiquitination degradation of MAVS protein,and to analyze the influenza virus under emotional stress load conditions.The biological mechanism of perceptual increase,improve the evaluation model and system of traditional Chinese medicine efficacy,and provide new targets for the study of antiviral mechanism of traditional Chinese medicine.First,in vivo and in vitro influenza virus susceptibility models were established using restraint stress-loaded mice and stress hormone CORT-loaded A549 cells,respectively.In the in vivo model,Kunming mice were randomly divided into 4groups:normal group,restraint stress group,virus group,and restraint stress loaded virus group.The mice in the restraint stress group and the restraint stress loaded virus group were given a continuous restraint stress load for 22 h.After 3 days,the 2×LD₅₀0 H1N1 virus was nasally instilled.After 5 days of infection,the lung tissues of the mice were taken.The results of qRT-PCR,Western blot and immunohistochemistry showed that the restraint stress load significantly increased the NP gene?p<0.001?and protein expression level in lung tissue after influenza infection in mice.The virus titer in the lung tissue of mice was detected by TCID₅₀assay.The results showed that the TCID₅₀0 value in the restraint stress loaded virus group was significantly higher than that in the virus group?p<0.01?.H&E staining and Giemsa staining of lung tissue sections showed that the inflammatory infiltration of the restraint stress virus group was worse than that of the virus control group,the number of inflammatory cells was increased,and the number of neutrophils and macrophages was increased.The above results indicate that restraint stress increases the susceptibility of mice to influenza virus.In the in vitro model,A549 cells were loaded with stress hormone CORT?100?M?for 48 h,then 10 TCID50 H1N1 virus was adsorbed for 2 h,and cultured for 12 h in the incubator.The results of qRT-PCR and Western blot showed that CORT could significantly increase the NP gene?p<0.001?and protein expression level in A549 cells after influenza virus infection.The TCID₅₀0 value of CORT load virus group was found significantly higher than the virus group?p<0.01?.Immunofluorescence results showed that the expression of NP protein was significantly higher than that of the virus group.The above experiments and data show that we have successfully established influenza virus susceptibility models in vivo and in vitro.MAVS-mediated IFN?secretion is an important pathway for host resistance to RNA virus infection.This study further utilized the established in vivo and in vitro susceptibility model to study the mechanism by which restraint stress/CORT affects the MAVS/p-IRF3/IFN?antiviral signaling pathway.Western blot results showed that restraint stress or CORT load significantly inhibited the expression of MAVS,p-IRF3 and IFN?protein induced by influenza virus?p<0.01?.Immunofluorescence results also confirmed a significant decrease in MAVS expression in the CORT-loaded virus group.However,qRT-PCR results showed that only IFN?gene expression was inhibited,MAVS mRNA levels did not change significantly,suggesting that MAVS protein reduction may be related to its degradation.To further investigate the degradation mechanism of MAVS protein,we used the protein synthesis inhibitor cycloheximide?CHX?and the proteasome inhibitor MG132 in"CORT+Poly I:C?dsRNA analog?"load A549 cells.It was shown that CHX significantly accelerated the degradation rate of MAVS,while MG132?10?M,12 h?increased the protein level of MAVS.On the other hand,we found that restraint stress or CORT increased the level of MAVS ubiquitination in lung tissue and A549cells of virus-infected mice.Therefore,we conclude that the reduction of MAVS protein caused by restraint stress/CORT is achieved by the ubiquitination degradation pathway.Finally,we studied and explored the degradation mechanism of MAVS ubiquitination induced by restraint stress/CORT.RNA-sequence results showed that restraint stress caused a significant up-regulation of mitochondrial fusion protein?Mitofusin2,Mfn2?,which was further confirmed by qPCR,Western blot and immunofluorescence experiments.Furthermore,we found that overexpression of the Mfn2 plasmid?pCDNA3.1-Mfn2-Flag?in A549 cells up-regulated the expression of NP after viral infection and down-regulated the expression of MAVS and IFN?;the results of si-Mfn2 assay were opposite to those of Mfn2 overexpression.Thus,we hypothesized that Mfn2 may be involved in the ubiquitination degradation mechanism of MAVS proteins.Co-IP experiments showed that restraint stress or CORT could increase the interaction between MAVS and Mfn2 in lung tissue of H1N1 infected mice and A549 cells.The results of immunofluorescence also showed that the colocalization of MAVS and Mfn2 was significantly increased.On the other hand,we found that overexpression of Mfn2 increased ubiquitination of MAVS in A549 cells and decreased MAVS protein levels,while si-Mfn2 gave the opposite result.To further confirm that Mfn2 is involved in the ubiquitination of MAVS,we transfected pCDNA3.1-MAVS-HA,pCDNA3.1-Mfn2-Flag,and pCMV-UB-Myc plasmids into HEK293T cells to establish a ubiquitination co-transfection system.It was found that the degree of ubiquitination of MAVS in the MAVS/Mfn2 and UB plasmid groups was significantly stronger than that in the group not transfected with Mfn2.Finally,in order to determine that Mfn2 promotes MAVS ubiquitination of E3ubiquitin ligase,we found that synovial protein 1?Synoviolin 1,SYVN1?with the highest scoring?Score=0.736?associated with MAVS ubiquitination was predicted by software?Ubibrowser?.Co-IP experiments showed that CORT significantly promoted the interaction of SYVN1 and MAVS in A549 cells.si-SYVN1significantly reduced ubiquitination of MAVS,increased MAVS and IFN?expression,and decreased NP protein expression,but there was no significant change in Mfn2 protein.The above various experimental techniques and methods have shown that CORT causes an increase in Mfn2,and Mfn2 recruits E3 ligase SYVN1 to promote ubiquitination degradation of MAVS.In summary,based on the previous research of our group,we further elucidated the biological mechanism of susceptibility to influenza virus induced by emotional stress using in vivo and in vitro influenza virus susceptibility models:CORT increased the expression of Mfn2,Mfn2 recruit E3 ligase SYVN1 triggered the ubiquitination of MAVS,which inhibited the MAVS/p-IRF3/IFN?antiviral signal,promoted viral replication,and increased the susceptibility of the body to influenza.The research results of this subject enrich the theory of emotionally ill Chinese medicine,and provide a new direction and angle for evaluating the efficacy and analytical mechanism of Chinese medicine and finding the target.