The Essential Role And Mechanism Of TRIM65 In MDA-5-mediated Antiviral Innate Immunity

Innate immune antiviral response is the first line of organism resistance to RNA virus,and RIG-1 like receptor mediated antiviral signaling activation is very important for it.RIG-I like receptor can sense RNA virus specifically,and then activate the downstream signaling pathway,which can lead to the production of IFN-I,as well as other cytokines.Resultantly,these cytokines can promote strong resistance to RNA virus,preventing heavy damage of organism.There are three members of RIG-I like receptor family,that are RIG-I,IDA-5 and LGP2.RIG-I and MDA-5 can sense different species of RNA virus respectively,while LGP2 can regulate the activation of the other two family members.Loss function of RIG-I and MDA-5 can make body susceptible to RNA virus,leading to many diseases and even death.In other aspect,the over activation of them also can cause the imbalance of homeostasis,leading to some autoimmune diseases,such as systemic lupus erythematosus.So the proper function of RIG-I and MDA-5 is critical for the resistance to RNA virus,as well as the homeostasis of organism.Currently,the mechanism of RIG-I activation is clear relatively.However,the mechanism of MDA-5 activation is poorly understood.Only we know is that phospholylation is important during MDA-5 activation,which is also critical to RIG-I activation.But whether there exit some other types of modification in this process is largely unknown.Especially whether ubiquitination,which is very important for RIG-I activation,is also essential for MDA-5 activation or not,as well as which E3 ubiquitin ligase participates in it needs to be investigated.According to these reasons,we studied the molecular mechanism of MDA-5 activation in this research.In our study,we identified the proteins associated with MDA-5 using co-inmunoprecipitation and mass spectrometry,and found that the E3 ligase TRIM65 had the highest number of matched peptides in the precipitates.According to our following results,we confirmed the strong and specific interaction between MDA-5 and TRIM65.To test whether this observed interaction plays an important role in MDA-5 activation,we constructed Trim65-/-mice.We infected the Trim65-/-mice or cells from them with MDA-5 specific agonists and found that the deletion of TRIM65 blocked MDA-5 activation.We also observed the similar results in human cells.As to the mechanism of TRIM65 regulatory role on MDA-5 activation,we found TRIM65 can promote MDA-5 K63 linked polyubiquitination at Lys743,and this promotion of ubiquitination can induce MDA-5 oligomerization,which is a key step for MDA-5 signal transduction.Our results found that TRIM65 is an essential factor of MDA-5 signaling pathway,and suggested that ubiquitination is critical to MDA-5 oligomerization and activation,as well as found a new target for MDA-5 associated diseases.