Innate immunity acts as the host’s first line of defense against viral invasion and viruses have evolved various strategies to evade host innate immunity to achieve infection. Toll-like receptors (TLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), NOD-like receptors (NLRs) and intracellular DNA sensors (cGAS, IFI16, et al.) are all well studied PRRs to detect viral RNA or DNA to trigger cellular signaling cascades, leading to the induction of type I interferons (IFNs), expression of IFN-stimulated genes (ISGs) and the establishment of antiviral state [17, 18]. As a DNA virus, innate immune-induced DNA sensing pathway plays a key role in controlling PRV infection, but a large number of studies have shown that PRV uses a variety of mechanisms to destroy the host's antiviral responses[19]. Yin et al. found that PRV inhibits type I and type Ⅲ interferon-induced signaling through proteasomal degradation of JAK1 and Tyk2 [20]. Kong et al. revealed the PRV tegument protein UL13 recruits RNF5 to inhibit STING-mediated antiviral immunity [5]. Lv et al. demonstrated that UL13 degrades PRDX1 to inhibit IFN production depending on its kinase activity [21]. More interesting, studies have discovered that RNA intermediates generated during DNA virus replication and host-derived RNAs induced by DNA viruses can also initiate the RLR-mediated signaling pathway. Recent studies have shown that UL13 of PRV inhibits RLR-mediated IFN-β production by suppressing the expression of RIG-I and MDA5 by inhibiting NF-κB transcription [6], which is similar with our findings that TRIM26 was recruited by PRV to negatively regulate the RLR signaling pathway. Previous studies have reported that IRF3 could be ubiquitinated and degraded by TRIM26 in the nucleus, suggesting that viral pathogens may induce the expression of TRIM26 to evade innate immunity [14]. In the present study, we found the expression of TRIM26 was upregulated significantly after PRV infection. Overexpression of TRIM26 inhibited the production of IFN-β, ISG15 and ISG56 triggered by poly (I:C) and SeV, conversely, knockdown of endogenous TRIM26 restored the production. However, Ran et al found TRIM26 was physically associated with TBK1, bridged TBK1-NEMO interaction and eventually mediated the activation of innate immune responses [9]. These conflicting reports implied that the role of TRIM26 is versatile and complicated, the underlining mechanism still need to be clarified and further studied.
Some alphaherpes viruses have developed effective ways to block RLRs-activated the MAVS-TBK1-IRF3 axis. For example, the VHS protein of HSV-2 decreased expression of RIG-I and MDA5 [22], Zhao et al identified UL37 of HSV-1 is a protein deamidase that targets RIG-I to block RNA-induced activation [23], Xing found that HSV-1 US11 binds to RIG-I and MDA-5 and inhibits their downstream signaling activation [24]. Given our previous results that TRIM26 was increased significantly after PRV infection and its positive role in regulating PRV infection. In the present study, we focused more on the mechanism that TRIM26 inhibited innate immune response. To initiate proper immune responses and avoid excessive harmful immune responses, the activities of MAVS are tightly and precisely regulated as the key component involved in RIG-I signaling pathway. For instance, SARS-CoV-2 ORF10 induces mitophagy-mediated MAVS degradation by binding to NIX to suppresses the antiviral innate immune response [25]. Senecavirus A-induced glycolysis facilitates virus replication by promoting lactate production that attenuates the interaction between MAVS and RIG-I [26]. TNKS1 and TNKS2 catalyzes the Poly (ADP-ribosyl) ation (PARylation) of MAVS at Glu137 residue, priming it for K48-linked polyubiquitination by the E3 ligase RNF146 and subsequent degradation [27]. E3 ligase TRIM25 has been showed that it interacts with ubiquitinated MAVS to be involved in type I interferon production after activation of the antiviral RIG-I-like receptors [28]. Interestingly, we found that TRIM26 interacted with MAVS by IP and confocal assay and decreased the expression of MAVS by western blot. The findings not only confirmed the negative role of TRIM26 in innate immune response as published before, but also expand our understanding of how DNA virus (PRV) use host proteins to antagonize RLR-mediated antiviral innate immune response.
Autophagy is a major degradative process mediated by autophagosome for breakdown of wrapped contents, which is considered to be nonselective when faced to cellular or environmental stresses in early studies [29]. However, more and more studies have indicated that autophagy selectively targets specific substrates through cargo receptors, especially in the crosstalk between viruses and host immune responses [30]. For instance, Tegument protein UL21 of alpha-herpesvirus inhibits the innate immunity by triggering CGAS degradation through TOLLIP-mediated selective autophagy [31]. Influenza A virus hijacks autophagy to recruit NBR1 to degrade MAVS through autophagosomes, inhibiting IFN response and promoting virus replication [32]. Porcine reproductive and respiratory syndrome virus utilized viral envelope (E) protein degrades DDX10 via SQSTM1/p62-dependent selective autophagy to antagonize the antiviral activity. Besides virus infection, iron metabolism-related gene HFE could also bind to MAVS for SQSTM1/p62-mediated MAVS degradation via selective autophagy [33]. Here, the TRIM26-mediated degradation of MAVS was mainly recovered by treatment with 3-MA and NH4Cl, but not MG132 or Z-VAD-FMK, indicating that autophagy-lysosome pathway might be involved in the process. In selective autophagy, p62/SQSTM1 acts as an important adaptor to identify specific organelles and protein aggregates and deliver them to autophagosomes for degradation. In recent years, considerable progress has been made in understanding the mechanisms underlying selective cargo phagocytosis in mammals, including the identification of ubiquitin-dependent selective autophagy receptors such as p62/SQSTM1, NBR1 (neighbor of BRCA1), OPTN (optineurin), and NDP52 (nuclear dot protein 52 kDa), which can bind both cargo and ubiquitin to initiate pathways leading to autophagy initiation and membrane recruitment [34, 35]. Our data indicated NDP52 interacted with both TRIM26 and MAVS, and TRIM26 induced MAVS degradation was almost entirely blocked in NDP52 knock down cells, demonstrating that TRIM26 degrades MAVS by promoting NDP52-mediated selective autophagy. Mounting evidence has shown ubiquitination is critical for recognizing cargos in selective autophagy, so whether the degradation in our study depends on the RING-finger domain of TRIM26 or the form of polyubiquitin chains linked to NDP52 and MAVS needs to be further studied.
Taken together, based on our study, the additional function of TRIM26 as a negative regulator in DNA virus-induced type I IFN signaling has been identified. We propose an important role for TRIM26 in antiviral immune responses, namely that after host infection with PRV, RIG-I sensing viral DNA activates RLR-mediated signaling, and TRIM26 is preferentially recruited by PRV and interacts with the selective autophagy receptor NDP52 to recognize ubiquitylated MAVS. NDP52 then delivers the MAVS to the autophagolysosome for degradation. TRIM26 down-regulates the protein abundance of MAVS, thereby inhibiting TBK1/IRF3 activation and antiviral immunity in negative feedback. Our study identified a novel regulatory mechanism by which TRIM26 evades the host antiviral immune response by triggering MAVS degradation via NDP52-mediated selective autophagy.