Molecular Mechanism Of Equine TRIM5α Activating AP-1 Signaling Pathway

TRIM5α which is a member of the tripartite motif family proteins consists of RING domain, B-box2 and coiled-coil domains that have referred to as RBCC proteins. The C-terminal SPRY domain confers retroviral capsid binding and specificity. TRIM5α, as a restriction factor, also trigger broad innate immune signaling for further viral suppression. Interestingly, equine TRIM5α(eqTRIM5α) that have not intact SPRY domain, lack capsid binding, but could restrict Equine infectious anemia virus(EIAV). The antiviral acivity of eqTRIM5α was evaluated in the HEK293 cell lines stably expressing eqTRIM5α established by lentiviral expressing system, furthermore eqTRIM5α can activate AP-1 and NF-κB pathways. This indicated that the regulation of cellular innate immune signaling pathways contribute to eqTRIM5α-mediated retrovirus restriction activity.The SPRY domain is necessary for primate TRIM5α activating innate immune signaling pathways in our previous reseach. However, eqTRIM5α contains only one fifth of full SPRY domain but still significantly activates AP-1 and NF-κB pathways, indicating a different antiviral mechanism may be used by eqTRIM5α. To determine the key amino acid of eqTRIM5α for activating AP-1 pathway, the eqTRIM5α truncated mutants(eqTRIM5-333, eqTRIM5-328, eqTRIM5-324, eqTRIM5-318, eqTRIM5-310, eqTRIM5-303, eqTRIM5-282, eqTRIM5-310-303, eqTRIM5-310-304, eqTRIM5-310-305, eqTRIM5-310-306, eqTRIM5-310-307, eqTRIM5-310-308, eqTRIM5-310-309), AP-1 and TK expressing plasmids were cotransfected to 293 T cells. The luciferase activities were detected after 24 h. Finally, we identified that the 303 Thr and 304 Leu are the key amino acids of eqTRIM5α for activating AP-1 and NF-κB pathways. To understand how eqTRIM5α activates AP-1 pathway, eqTRIM5α/mutants were cotransfected with TAB2/TAK1 to evaluate their interaction, our results indicated that TAB2 could be degraded by eqTRIM5α and eqTRIM5α mutants. But eqTRIM5α mutants that can not activate AP-1 pathway also can not interact with TAK1. The eqTRIM5α/eqTRIM5α mutants were tested for the ability to catalyze the ubiquitination of TAK1. The results revealed that eqTRIM5α may activate TAK1 by stimulating the ubiquitination of TAK1, which further activate AP-1 and NF-κB pathways.In summary, our research revealed the molecular mechanism of eqTRIM5α activated AP-1 signaling pathway and discovered the important role of eqTRIM5α activating innate immune signaling in antiretroviral activity. This would contribute to understand the coevolution of host restriction factors and retroviruses. The eqTRIM5α has potential to be a antiviral model to research its application prospect.