| Influenza A viruses(IAVs)are contagious pathogens that cause acute respiratory inflammation and can infect a broad variety of animals,including mammals and birds.Occasional outbreak and annual seasonal or regionnal epidemics of IAVs pose a great threat to the health of humans and animals,and also result in huge economic losses.The interaction between IAV and host is extremely complicated,in which a large number of host factors are involved.Therefore,addressing the roles of these host factors in IAV infection is of great significance to further understand the pathogenesis of IAV.Previous studies showed that after IAV entry into the host cell,viral RNA can be recognized by host pattern recognition receptors(PRRs),which induces the secretion of interferons(IFNs)and other cytokines like IL6.These cytokines subsequently activate the JAK-STAT signaling pathway by binding to their specific receptors,which leads to the expression of considerable effector molecules,such as interferon stimulating genes(ISGs).The effector molecules exert a directly antiviral influence.In the STAT family,STAT1 and STAT2 have been demonstrated to play crucial roles in antiviral responses.However,little is known about the role and activation rule of STAT3 during the IAV infection.It is considered traditionally that the virus infection first leads to the production of cytokines,which can activate STAT proteins subsequently.However,we found that in the early stage of IAV infection,activation of STAT3 arises before the production of cytokines such as IL6.Therefore,we speculated that the early phosphorylation of STAT3 at 705 induced by IAV was independent of cytokines,including IL6.Thus,we used the supernatant of IAV infected A549 cells to stimulate fresh A549 cells,and found that supernatant of the cells infected for 3h and 4h was unable to phosphorylate STAT3 at 705.Subsequently,we knocked down the gene expression of interleukin 6 receptor(IL6R)and co-receptor(gp130)in A549 cells using specific shRNA,and found that the phosphorylation level of STAT3 at 705 at 4 hour after IAV infection did not change obviously,but significantly decreased at 12 hour post-infection.In addition,phosphorylation of STAT3 at 705 was also comparable between the IL6 knock-out mice and wild-type mice at 4 hour after IAV infection.Further study demonstrated that the early activation of STAT3 at 705 induced by IAV mainly depended on the RIG-I/MAVS signaling pathway,but was not affected by other PRRs such as MDA5,TLR3,TLR7 and transcription factors such as NF-κB,IRF3 and IRF7.Together,these data indicated that the phosphorylation of STAT3 at tyrosine 705 in the early stage of IAV infection is independent of cytokine including IL6,but depends on RIG-Ⅰ/MAVS signal pathway directly.Importantly,we found that knocking down the STAT3 expression in A549 cells with specific shRNA can significantly enhance the replication of influenza virus.Moreover,a vector carrying a dominant negative mutant of STAT3(pNL-STAT3 Y705F)was constructed and expressed in A549 cells.We observed that forced expression of STAT3-Y705F notably promoted IAV replication compared with the control group.Additionally,animal experiments showed that heterozygous mice with STAT3 705 tyrosine site mutation(STAT3-Y705F)also showed lower survival,higher lung viral load and more sever lung injury after viral infection as compared to wild type mice.These results proved that the activation of STAT3 705 tyrosine site induced by IAV played a prominent role in antiviral response.In summary,our results indicte that 1)The activation of STAT3 in early stage of IAV infection is independent of cytokines such as IL6,but related to the RIG-I/MAVS pathway.2)Phosphorylation of STAT3 at 705 in host cells may play a crucial role in inhibiting viral replication during IAV infection.These data provide a better understanding of the mechanism underlying interaction between host factors and influenza A viruses,and provide novel scientific evidence for revealing the pathogenesis of influenza A virus. |
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