Negative Regulation of Interferon-β Production by Alternative Splicing of Tumor Necrosis Factor Receptor-Associated Factor 3 in Ducks

Abstract

Tumor necrosis factor receptor-associated factor 3 (TRAF3), an intracellular signal transducer, is identified as an important component of Toll-like receptors and RIG-I-like receptors induced type I interferon (IFN) signaling pathways. Previous studies have clarified TRAF3 function in mammals, but little is known about the role of TRAF3 in ducks. Here, we cloned and characterized the full-length duck TRAF3 (duTRAF3) gene and an alternatively spliced isoform of duTRAF3 (duTRAF3-S) lacking the fragment encoding amino acids 217–319, from duck embryo fibroblasts (DEFs). We found that duTRAF3 and duTRAF3-S played different roles in regulating IFN-β production in DEFs. duTRAF3 through its TRAF domain interacted with duMAVS or duTRIF, leading to the production of IFN-β. However, duTRAF3-S, containing the TRAF domain, was unable to bind duMAVS or duTRIF due to the intramolecular binding between the N- and C-terminal of duTRAF3-S that blocked the function of its TRAF domain. Further analysis identified that duTRAF3-S competed with duTRAF3 itself for binding to duTRAF3, perturbing duTRAF3 self-association, which impaired the assembly of duTRAF3-duMAVS/duTRIF complex, ultimately resulted in a reduced production of IFN-β. These findings suggest that duTRAF3 is an important regulator of duck innate immune signaling and reveal a novel mechanism for the negative regulation of IFN-β production via changing the formation of the homo-oligomerization of wild molecules, implying a novel regulatory role of truncated proteins.

Innate immune responses to duck Tembusu virus infection

“…Increasing intracellular signal transducers were identified, numerous studies showed that many proteins could be involved in DTMUV infection. For example, the TRIF in duck embryo fibroblasts (DEF) cells infected with DTMUV was upregulated [77], duck TRAF3 can interact with the upstream molecule TRIF, and leading to the production of IFN-β, overexpression of TRAF3 inhibited the replication of DTMUV [78], while the overexpression of duck TBK1 and IRF7 dramatically reduced the replication of DTMUV in DEF cells [79][80][81]. The results above clearly demonstrated that a TLR3mediated signaling pathway was essential for defending against DTMUV infection.…”

Pattern Recognition Receptor Signaling and Innate Responses to Influenza A Viruses in the Mallard Duck, Compared to Humans and Chickens

“…In chicken embryonic fibroblasts (CEF) cells, TRAF3 (chTRAF3) is upregulated in response to poly (I:C) stimulation, NDV infection and poly dA-dT, suggesting it is important in both DNA and RNA viral infections (Yang et al, 2015). Similarly, duTRAF3 is also upregulated in DEF cells stimulated with poly (I:C) and the authors also found that overexpression of duTRAF3 could control both IAV and duck Tembusu virus replication (Wei et al, 2018).…”

“…TRAF3 is most highly expressed in lung, spleen, and thymus of 2-week-old chickens (Yang et al, 2015). Duck TRAF3 (duTRAF3) however, has a uniform expression pattern with only slightly higher amounts of TRAF3 expression seen in the brain, and the lowest levels in the lung (Wei et al, 2018). In chicken embryonic fibroblasts (CEF) cells, TRAF3 (chTRAF3) is upregulated in response to poly (I:C) stimulation, NDV infection and poly dA-dT, suggesting it is important in both DNA and RNA viral infections (Yang et al, 2015).…”

“…Curiously, a truncated version of duTRAF3 was also found, named duTRAF3-S (splice isoform duck TRAF3) (Wei et al, 2018). This splice variant is missing key N-terminal catalytic domains but can still bind to both TBK1 and MAVS with its Cterminal TRAF domain.…”