Abstract
The primary role of the innate immune response is to limit the spread of infectious pathogens, with activation of Toll-like receptor (TLR) and RIG-like receptor (RLR) pathways resulting in a pro-inflammatory response required to combat infection. Limiting the activation of these signaling pathways is likewise essential to prevent tissue injury in the host. Triad3A is an E3 ubiquitin ligase that interacts with several components of TLR signaling and modulates TLR activity. In the present study, we demonstrate that Triad3A negatively regulates the RIG-I RNA sensing pathway through Lys48-linked, ubiquitin-mediated degradation of the tumor necrosis factor receptor-associated factor 3 (TRAF3) adapter. Triad3A was induced following dsRNA exposure or virus infection and decreased TRAF3 levels in a dose-dependent manner; moreover, Triad3A expression blocked IRF-3 activation by Ser-396 phosphorylation and inhibited the expression of type 1 interferon and antiviral genes. Lys48-linked ubiquitination of TRAF3 by Triad3A increased TRAF3 turnover, whereas reduction of Triad3A expression by stable shRNA expression correlated with an increase in TRAF3 protein expression and enhancement of the antiviral response following VSV or Sendai virus infection. Triad3A and TRAF3 physically interacted together, and TRAF3 residues Y440 and Q442—previously shown to be important for association with the MAVS adapter—were also critical for Triad3A. Point mutation of the TRAF-Interacting-Motif (TIM) of Triad3A abrogated its ability to interact with TRAF3 and modulate RIG-I signaling. TRAF3 appears to undergo sequential ubiquitin “immuno-editing” following virus infection that is crucial for regulation of RIG-I-dependent signaling to the antiviral response. Thus, Triad3A represents a versatile E3 ubiquitin ligase that negatively regulates RIG-like receptor signaling by targeting TRAF3 for degradation following RNA virus infection.
Highlights
Upon recognition of specific molecular components of viruses, the host cell activates multiple signaling cascades that stimulate an innate antiviral response, resulting in the disruption of viral replication, and the mobilization of the adaptive arm of the immune system
Our study demonstrates that the E3 ubiquitin ligase Triad3A negatively regulates the retinoic acid-inducible gene-I (RIG-I)-like receptor pathway by targeting the adapter molecule tumor necrosis factor receptor-associated factor 3 (TRAF3) for proteasomal degradation through Lys48-linked ubiquitin-mediated degradation
A dose-response curve was performed using the I Signaling response element (ISRE) promoter with increasing amounts of Triad3A and DRIG-I, mitochondrial antiviral signaling (MAVS), TIR-domaincontaining adapter-inducing IFN-b (TRIF), or TBK1 expression plasmids; DRIG-I resulted in 893-fold induction of the ISRE promoter, and Triad3A coexpression diminished activation in a dose dependent manner (Figure S1A)
Summary
Upon recognition of specific molecular components of viruses, the host cell activates multiple signaling cascades that stimulate an innate antiviral response, resulting in the disruption of viral replication, and the mobilization of the adaptive arm of the immune system. RIGI-like receptors (RLRs) - the retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene-5 (MDA-5) - are novel cytoplasmic RNA helicases that recognize viral RNA present within the cytoplasm. Both TLR7 and TLR9 are critical for recognition of viral nucleic acids in the endosomes of plasmacytoid dendritic cells (pDCs), most other cell types recognize viral RNA intermediates through the RLR arm of the innate immune response [4,5,6]. The C-terminal regulatory domain (CTD) (aa 792–925) of RIG-I binds viral RNA in a 59-triphosphatedependent manner and activates RIG-I ATPase inducing RNAdependent dimerization and structural alterations that enable the CARD domain to interact with other downstream adapter protein(s) leading to the transcription of antiviral genes [7,8,9]
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