Enterovirus713C Antagonizes Innate Immunity Through Cleavage Of The TAK1Complex

Since Hand-foot-and-mouth disease (HFMD) has been listed as a category C infectious disease of China in2008, its reported incidences and deaths of HFDM have ranked first in all category C infectious disease. HFMD, which seriously endangers the children’s health, harms the economic development and disrupts the social stability, has become the priority of disease control and prevention in China. Enterovirus71(EV71) is the major causative pathogen of HFMD, but its pathogenicity has not been fully understood. Previous studies indicate that innate immune evasion plays a key role in EV71infection. We have reported that EV71antagonized host innate immunity by blocking the innate immune signaling pathways, but its mechanisms remain unclear. Here, we show that another innate immune signaling factor, TGF-beta activated kinase1(TAK1), is cleaved by EV713C. Additional work has been applied to investigate the mechanisms of influence and regulation between EV713C and TAK1complex based on this discovery.EV71infected cells can reduce the expression of TAKl complex, including TAK1, TAK1binding protein1,2, and3(TAB1, TAB2and TAB3). Results of immune coprecipitation show that3C interacts with TAK1and TAB2, meanwhile3C fails to cause the activation of NF-κB promoter by mediating cleavage of TAK1complex (TAK1,TAB1,TAB2and TAB3). By testing the effect of the3C protease inhibitor and3C mutants toward TAK1protein complexes, we found that in presence of3C protease inhibitor,3C fails to mediate cleavage of TAK1complexes. Similarly,3C mutants with abolished protease activity sites are incapable to cut TAK1complexes, revealing that the protease activity of3C is essential for cleavage of TAK1complex. Using dual-luciferase report system, we demonstrate that3C mutants fail to restrain the activation of NF-κB promoter induced by TAK1complex thus further illustrate the importance of3C protease in TAK1-mediated signaling pathways. We analyzed a series of suspectable cleavage sites of TAK1complex by inducing mutations. The3C protein targets TAB2at Q113-S114, TAK1at Q360-S361, TAB1at both Q414-G415and Q451-S452and TAB3at both Q173-G714and Q343-G344. Besides, overexpression of TAB2inhibits EV71replication, whereas addition of cleaved fragments of TAB2does not lead to such effect. These results suggest that the balance between TAK1complex and3C may be an important control point in virus infection.In addition, EV71infection can down-regulates the expression of TRAF3(an critical adaptor of TLR.3signaling pathways) by3C-mediated cleavage of TRAF3(the cleavage site is Q242-S243).3C also reduces interaction between TRIF and TRAF3, which may in turn antagonize type I interferon response.To sum up, our study identified new targets of EV71’s antagonism to host innate immune response, which delineated the complexity of immune-evasion mechanisms of EV71and further enriched the understanding of EV71’s antagonism mechanisms of host innate immunity, and provided the theoretical basis for the elucidation of pathogenesis and the discovery of antiviral drug targets of EV71.