Regulation Mechanism Of NF-κB Activation By Enterovirus 71 2C Protein

Enterovirus71(EV71) is one of primary pathogens that leads to hand-foot-mouth disease, and also serious nervous system symptoms consist of encephalitis, aseptic meningitis, neurogenic pulmonary edema. As a member of picornaviridae family, EV71with a7.4kb positive-strand RNA genome, encodes a polyprotein, which is cleaved to generate4structural proteins VP1, VP2, VP3, VP4and7non-structural proteins2A,2B,2C,3A,3B,3C,3D.EV712C protein is composed of329aa and shares97.5%and63.3%protein sequence identity with coxsackievirus A16and poliovirus, respectively. NF-κB plays a key role in host defend against virus infection. p65/50heterodimer is the most abundant member of NF-κB family. Under the physiologically resting state of host cell, p65/50binding with inhibitory protein IκBα/β forms inactive trimer and resides in cytoplasm. Diverse set of stimuli could stimulate IκB-a/β phosphorylation and ubiquitination. Ubiquitinated IκBα is directed to proteasome for degradation, and thus p65/p50dimer is released and activated. Active p65/p50dimer translocates from cytoplasm to nuclear, binds with specific DNA sequence and promotes downstream genes transcription, then such as cytokines, chemokines, enzymes and adhesion molecules, which are critical for host defending virus infection through innate immunity and adaptive immunity responses. A member of viruses including cowpox, coxsackievirus, hepatitis C virus, HIV-1, poliovirus and SARS-coronaviruscan can manipulate NF-κB pathway.To explore the physiological function of2C, we screened the potential partners for2C using the yeast two-hybrid (Y2H) approach. One of candidate proteins is p65, a member of NF-κB family. It is reported that EV712C protein inhibits TNF-a-mediated activation of NF-κB by suppression IKKβ phosphorylation. We validated the interaction between2C and p65through co-immunoprecipitation and GST pull down experiments. In addition, using confocal laser microscopy, we found that2C co-localized with p65. To map the region of p65responsible for association with2C, we truncated p65and found that the minimal region for association with2C is IPT domain, which dimerizes p65and p50. We found that2C competed with p50for association with p65and inhibited p65/p50dimerization. To map the region of2C for association with p65IPT domain, we truncated2C and found that2C105-125aa and126-263aa can bind to p65IPT domain. We also verified that both of2C105-125aa and126-263aa inhibited NF-κB activation. We found that2C truncations1-104aa,1-113aa,1-117aa,1-121aa,1-125aa inhibited NF-κB activation. It is reported that2C1-125aa interacted with IKKβ and inhibited IKKβ phosphorylation. We found IKKβ interacted with2C1-104aa and105-125aa,2C105-121aa interacted with IKKβ but not p65IPT domain.Taken together, we identified the2C-p65interaction and demonstrated that EV712C protein inhibited NF-κB activation by suppressing p65/p60dimerization, which will shed further mechanistic insights into antiviral innate immunity by EV71.