The Molecular Mechanism Of TRIM35 Protein Antagonizing Influenza Virus Infection

Influenza A virus?IAV?is an enveloped virus with a segmented genome consisting of eight single-stranded,negative-sense RNAs.The worldwide prevalence of IAV has caused major public health security problems and posed severe threat to the survival and development of humans.Like all other viruses,IAV interacts directly or indirectly with a large number of host proteins in the virus life cycle.Identifying host factors that play a promoting or inhibitory role in the process of virus replication can provide valuable new targets for the development of antiviral drugs.Tripartite motif containing?TRIM?proteins have a variety of different functions,and participate in various biological processes such as cell differentiation,apoptosis,transcriptional regulation and signal transduction.TRIM proteins are also important effectors for the host innate immune system to defend against virus infection.In order to study the regulatory effect of TRIM family proteins on IAV replication,we detected the effects of 30 different TRIM family proteins on IAV propagation by virus plaque titration assay.We found that several TRIM family proteins possessed the anti-IAV activity,and selected TRIM35 for further study.Using IFN-?and ISRE promoter reporter assay system,we verified that TRIM35 played a positive regulatory role in innate immune signaling pathway.It was shown that overexpression of TRIM35 inhibited IAV replication,while siRNA knockdown of TRIM35 expression or knockout of TRIM35 expression promoted influenza virus replication.In addition,we established a model of Trim35^-/-mice.After infection with A/WSN/33?H1N1?virus,in comparison with the wild-type control group,the weight of Trim35^-/-mice decreased more significantly until death,while all the wild-type mice survived.The virus replication titers in the lungs of Trim35^-/-mice were significantly higher than those of the wild-type mice,and more severe bronchopneumonia with more prominent viral antigen expression was observed in the lungs of Trim35^-/-mice compared with wild-type mice.By screening of the key molecules in the RIG-I signaling pathway,we identified TRAF3 as the target protein of TRIM35,and verified the interaction between them by co-immunoprecipitation?co-IP?experiments.We constructed a series of truncation mutants of TRIM35 and TRAF3,and found that the key domains responsible for their binding are the PRY/SPRY domain of TRIM35 and the RING domain of TRAF3.Notably,we found that TRIM35 played a positive role in regulating the RIG-I signaling pathway through catalyzing K63-linked ubiquitination of TRAF3,and two cysteine residues at postion36 and 60?C36,C60?of TRIM35 are the key sites for mediating K63-linked ubiquitination of TRAF3.On the other hand,we found that among the 10 major proteins of IAV,only PB2 protein interacted with TRAF3,and PB2 inhibited the transduction of RIG-I signaling pathway and the formation of VISA-TRAF3 complex by blocking the K63-linked ubiquitination of TRAF3.Finally,we found that TRIM35 interacted with IAV PB2,and catalyzed K48-linked ubiquitination of PB2,resulting in proteasomal degradation of PB2 and inhibited viral ribonucleoprotein complex?vRNP?activity and virus replication.Further study showed that the RING domain of TRIM35 is the key domain in exerting antiviral activity,and the amino acid at position 736 of PB2 is the key acceptor site of TRIM35-meidated K48-linked ubiquitination.In summary,our in vitro and in vivo experiments revealed that through dual mechanisms,TRIM35protein played novel roles in RIG-I-mediated innate immunity and defense against IAV infection,which provides potential clues for the development of anti-influenza drugs.