Binding of Duck Tembusu Virus Nonstructural Protein 2A to Duck STING Disrupts Induction of Its Signal Transduction Cascade To Inhibit Beta Interferon Induction

Abstract

Duck Tembusu virus (DTMUV), which is similar to other mosquito-borne flaviviruses that replicate well in most mammalian cells, is an emerging pathogenic flavivirus that has caused epidemics in egg-laying and breeding waterfowl. Immune organ defects and neurological dysfunction are the main clinical symptoms of DTMUV infection. Preinfection with DTMUV makes the virus impervious to later interferon (IFN) treatment, revealing that DTMUV has evolved some strategies to defend against host IFN-dependent antiviral responses. Immune inhibition was further confirmed by screening for DTMUV-encoded proteins, which suggested that NS2A significantly inhibited IFN-β and IFN-stimulated response element (ISRE) promoter activity in a dose-dependent manner and facilitated reinfection with duck plague virus (DPV). DTMUV NS2A was able to inhibit duck retinoic acid-inducible gene-I (RIG-I)-, and melanoma differentiation-associated gene 5 (MDA5)-, mitochondrial-localized adaptor molecules (MAVS)-, stimulator of interferon genes (STING)-, and TANK-binding kinase 1 (TBK1)-induced IFN-β transcription, but not duck TBK1- and interferon regulatory factor 7 (IRF7)-mediated effective phases of IFN response. Furthermore, we found that NS2A competed with duTBK1 in binding to duck STING (duSTING), impaired duSTING-duSTING binding, and reduced duTBK1 phosphorylation, leading to the subsequent inhibition of IFN production. Importantly, we first identified that the W164A, Y167A, and S361A mutations in duSTING significantly impaired the NS2A-duSTING interaction, which is important for NS2A-induced IFN-β inhibition. Hence, our data demonstrated that DTMUV NS2A disrupts duSTING-dependent antiviral cellular defenses by binding with duSTING, which provides a novel mechanism by which DTMUV subverts host innate immune responses. The potential interaction sites between NS2A and duSTING may be the targets of future novel antiviral therapies and vaccine development. IMPORTANCE Flavivirus infections are transmitted through mosquitos or ticks and lead to significant morbidity and mortality worldwide with a spectrum of manifestations. Infection with an emerging flavivirus, DTMUV, manifests with clinical symptoms that include lesions of the immune organs and neurological dysfunction, leading to heavy egg drop and causing serious harm to the duck industry in China, Thailand, Malaysia, and other Southeast Asian countries. Mosquito cells, bird cells, and mammalian cell lines are all susceptible to DTMUV infection. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and may pose a threat to mammalian health. However, the pathogenesis of DTMUV is largely unclear. Our results show that NS2A strongly blocks the STING-induced signal transduction cascade by binding with STING, which subsequently blocks STING-STING binding and TBK1 phosphorylation. More importantly, the W164, Y167, or S361 residues in duSTING were identified as important interaction sites between STING and NS2A that are vital for NS2A-induced IFN production and effective phases of IFN response. Uncovering the mechanism by which DTMUV NS2A inhibits IFN in the cells of its natural hosts, ducks, will help us understand the role of NS2A in DTMUV pathogenicity.

Innate Immune DNA Sensing of Flaviviruses

“…Furthermore, they found that DTMUV NS2B3 cleaves duck STING (duSTING) through an NS2B dependent manner and that the binding of NS2B with duSTING distal tail (residue 221-225) is a prerequisite for the subsequent cleaving by NS2B3 complex. Moreover, in their latest work, they discovered that DTMUV NS2A competes with duck TBK1 (duTBK1) to bind with duSTING, thus disrupting duSTING dimerization and inhibiting downstream IFN production [88]. The work they performed using an avian model will undoubtedly inform the work we do in mammalian systems in the future and could potentially provide evidence for a conserved pan-flavivirus strategy of modulating host innate immune space by targeting the cGAS/STING pathway [138,139].…”

“…Although the focus of this review is on the mammalian cytosolic DNA sensory system and the flaviviral strategies on regulation and modulation of those pathways, the authors believe it is noteworthy to mention some elucidating new research coming from the field of avian flavivirus (duck Tembusu virus, or DTMUV) [88,89,137].…”

Innate immune responses to duck Tembusu virus infection

“…The NS2A protein and TBK1 competed binding to duck STING, disrupted STING dimer formation and reduced TBK1 phosphorylation, leading to the inhibition of IFN-β production. The amino acid residues at 164, 167, and 361 in STING were important for NS2A binding with STING [145]. However, NS2B3 polyproteins inhibited IFN induction by hydrolyzing duck STING, and further mapping analysis showed the scissile bond located between the R84 and G85 residues of STING [146].…”

Amelioration of Beta Interferon Inhibition by NS4B Contributes to Attenuating Tembusu Virus Virulence in Ducks

“…Additionally, in our previous study, we analyzed the ability of the 10 proteins encoded by duck TMUV to block the IFN system and found that the expression of NS2A, NS2B, and 2KNS4B resulted in robust IFN signaling inhibition. We further found that duck TMUV NS2A inhibited IFNb induction by targeting duck STING (13), and duck TMUV NS2B3 could cleave and bind duck STING to subvert the induction of IFNb (14), suggesting that different duck TMUV proteins could commonly target the same cellular components through different strategies to suppress IFNb production. However, the mechanism by which duck TMUV NS4B subverts the host innate immune response is unclear and requires further study.…”

“…The sequence of the 2KNS4B gene was amplified from the duck TMUV CQW1 strain genome and cloned into the pCAGGS expression vector with a His tag at the C terminus using standard molecular biology techniques. The plasmids pCAGGS-duRIG-I, duMDA5, duMAVS, duTBK1, duIRF7, and duSTING-Flag and pBiT-LgBiT-duRIG-I, duMDA5, duMAVS, duTBK1, duIRF7, duSTING-Myc, pBiT-SmBiT-duTBK1 and STING-Flag were constructed as described in our previous study (13) for the NanoLuc Binary Technology (NanoBiT) assay. 2KNS4B and its truncations or mutants were cloned into pBiT2.1C-SmBiT with a Flag tag.…”