The cGAS-mediated innate immune response forms the first line of defense that protect hosts from invasion by DNA viruses. After virus infection, the IFN-β signaling pathway is activated to induce IFN-β and ISGs expression, thereby initiating the appropriate adaptive immune response. Investigating the mechanisms underlying the innate immune response holds great potential for bettering disease control and designing effective vaccines. In the present study, we first investigated the roles of STRAP in type I IFN-mediated innate immunity response against PRV. The overexpression of STRAP exhibited a notable inhibition effect on the activation of IFN-β promoter and IFN-β induction in response to PRV, whereas STRAP knockdown had the opposite effects. This finding establishes a critical role for STRAP in the innate immune response.
While much is known about STRAP as a scaffold protein implicated in diverse cellular functions, its involvement in the regulation of innate immunity remains poorly understood [18, 19, 34, 35]. In this study, we provided five lines of evidence indicating that STRAP exerts a positive regulatory effect on the type I IFN signaling response to PRV infection. Firstly, we observed a significant upregulation of STRAP expression in response to PRV, suggesting a potential crucial role for STRAP during PRV infection. Secondly, we demonstrated that overexpression or silencing of STRAP results in heightened or diminished production of IFN-I triggered by PRV infection, respectively, underscoring the critical role of STRAP in promoting the innate immune response against PRV. Thirdly, we uncovered that STRAP facilitates the IFN-I signaling pathway against PRV infection by targeting the kinase TBK1. Fourthly, we revealed that both CT and WD40 7 − 6 domains contribute to STRAP’s function in the IFN-I signaling pathway. Lastly, we showed that STRAP impairs the ability of the PRV-UL50 to degrade TBK1 expression, thereby promoting the interaction between STRAP and TBK1. Together, these findings establish STRAP as a positive regulatory in IFN-I signaling and highlight its significance in host innate immunity against PRV infection, potentially extending to other viral infections.
Notably, previous studies have shown that STRAP positively regulates the TLR-mediated signaling pathway [27], but negatively regulates the TGF-β signaling pathway [21]. Here, we identified that STRAP functions as a positive regulator in the IFN-I signaling pathway and participates in host antiviral response against PRV. Again, STRAP exerts its antiviral activity via interacting with TBK1. STRAP has been shown to interacting with PDK1 and p53 to regulate ASK1 and p53 function [20, 25]. It has been reported that STRAP directly interacts with Smad proteins and suppresses TGF-β signaling [21]. Therefore, it is likely that STRAP functionally links TBK1 and ASK1, TGF-β, p53, PI3K and IFN-I signaling pathways. Furthermore, we observed that STRAP is predominantly distributed in the cytoplasm, with only a small proportion in the nucleus (Fig. 1D). Thus, it can be inferred that STRAP functions in the cytoplasm.
WD40 repeat proteins appear to severe regulatory functions in various cellular processes [30]. The WD40 domains of STRAP play a critical role in mediating protein-to-protein interactions, despite lacking intrinsic enzymatic activity. Our data provides evidence that the WD40 domains of STRAP play a crucial role in the interaction between STRAP with TBK1, as well as in its antiviral response. To investigate the significance of WD40 region, we constructed four truncations of STRAP by selectively deleting one or two WD40 repeats, with or without intervening regions, from the C terminus. In comparison to the wild-type and C-terminal deleted STRAP, four truncates exhibited the lack of interaction with TBK1 and anti-PRV activity. It is plausible that both the CT and WD40 7 − 6 domains of STRAP play a critical role in recruiting other cellular proteins in IFN-I signaling. This regulation is comparable to the synergistic effect of STRAP-Smad7 interaction in the suppression of TGF-β signaling [21], in line with STRAP’s positive role in regulating the MyD88-dependent TLR2/4 signaling pathway [27]. However, in contrast to our results, one previous study found that the C-terminal domain is required for its functional activity in TLR3-mediated cytokines production [36]. In conclusion, our research reveals a previously unidentified role for STRAP in host defense against PRV infection.
TBK1, a key kinase for IFN production, undergoes phosphorylation after virus infection, which is a required step for its activation as well as type I IFNs production [10]. The data presented in this study provide evidence that overexpression of STRAP enhanced TBK1 phosphorylation, and STRAP knockdown leads to a decrease in TBK1 phosphorylation following PRV infection (Fig. 3E). This supports the crucial involvement of STRAP in TBK1 activation to facilitate the IFN-I signaling. However, the precise mechanism underlying how STRAP regulates the kinase activity of TBK1 requires further investigation. Additionally, the stability of TBK1 is also essential for its function in modulating type I IFN signaling. TBK1 can be degraded through ubiquitin-proteasome pathway by many regulators, such as DTX4, NLRP4, TRIM27, USP38, TRIP and TRAF3IP3 [37–41]. We demonstrated that UL50 encoded by PRV possesses the capability to induce TBK1 degradation through both the proteasome and autophagy pathways. Notably, the TBK1 degradation induced by PRV-UL50 was restored by STRAP overexpression. Thus, STRAP might play essential roles in the maintenance of TBK1 stability.
To effectively establish and sustain infection, herpesviruses, including HCMV and HSV-1, have evolved diverse mechanisms to circumvent host antiviral immunity and promote viral infection. However, research on PRV proteins involved in modulation of the cGAS-STING signaling pathway remains scarce compared to other herpesviruses [12, 14, 15]. We found that STRAP can interact with PRV-UL50, a tegument protein encoded by PRV. Additionally, we also identified that UL50 degraded TBK1 expression, thereby impairing the phosphorylation of IRF3, which supports that PRV inhibit the type I IFN signaling to establish persistent infection. Significantly, our findings identified that STRAP exhibits competitive interacting with TBK1, results in the disruption of STRAP-UL50 interaction, and enhances TBK1 stability, subsequent promoting production of IFN-I. These findings not only provide further evidence regarding the regulatory mechanism of STRAP on the IFN-I signaling pathway, but also present a proposed mechanism through which UL50 inhibits IFN-I production. This argument offers a more comprehensive explanation for why STRAP promotes cellular antiviral activity in response to PRV.
Based on our findings, we proposed a model elucidating the role of STRAP in antiviral innate immune reposes (Fig. 9). STRAP positively regulated PRV-triggered innate immune response. STRAP interacts with TBK1 and impedes the degradation of TBK1 induced by PRV-UL50, resulting in enhanced production of IFN-I and its downstream ISGs, inhibiting PRV replication. The CT and WD40 7 − 6 domains of STRAP are responsible for its function. In conclusion, our study revealed an underlying mechanism of how STRAP positively regulated type I IFN signaling by targeting TBK1, which would contribute to understanding the positive regulation of host innate immune responses and the function of STRAP during PRV infection.