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Association of ZAP and TRIM25 RNA binding mutants with SINV RNA. (A, B) ZAP KO or (C) TRIM25 KO 293T cells were transfected with (A) ZAP zinc finger (ZnF) mutants, (B) ZAP CpG RNA binding cavity mutants, or (C) TRIM25 mutants. ZAP or TRIM25 pulled down (IP) with Sindbis virus (SINV) or firefly luciferase (Fluc) RNA and in whole cell lysate (WCL) were assayed by immunoblot (IB). Blots were quantified with ImageJ. Data are representative of two independent experiments.
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The innate immune response controls the acute phase of virus infections; critical to this response is the induction of type I interferon (IFN) and resultant IFN-stimulated genes to establish an antiviral environment. One such gene, zinc finger antiviral protein (ZAP), is a potent antiviral factor that inhibits replication of diverse RNA and DNA vir...
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... Previous mutagenesis studies have revealed that the RNA binding of both ZAP and TRIM25 is important for their restriction of viral replication, particularly for mutation in the CpG binding site of ZAP [10,98]. Secondly, the interaction of ZAP with TRIM25 is critical for their inhibition on viral protein translation, and TRIM25 is required for ZAP-mediated antiviral responses [98,99]. To confirm the contribution of TRIM25 to ZAP RNA binding, mutants of the TRIM25 RNA-binding domain SPRY and SPRY/7K motif were constructed, and the results indicated that the loss of TRIM25 RNA-binding ability greatly reduces ZAP antiviral activity [14]. ...
... Further experiments unveiled that TRIM25-mediated proteasomal degradation is crucial for the maintenance of normal levels of ZAP in the host environment [10]. The interaction between ZAP and TRIM25 may initiate the recruitment of TRIM25, which enhances the ubiquitination and activation of the cellular regulatory substrates that inhibit viral mRNA expression, particularly during JEV infection [99]. ...
Tripartite motif (TRIM) 25 is a member of the TRIM E3 ubiquitin ligase family, which plays multiple roles in anti-tumor and antiviral defenses through various pathways. Its RBCC and SPRY/PRY domains work cooperatively for its oligomerization and subsequent activation of ligase activity. TRIM25 expression is regulated by several proteins and RNAs, and it functionally participates in the post-transcriptional and translational modification of antiviral regulators, such as RIG-I, ZAP, and avSGs. Conversely, the antiviral functions of TRIM25 are inhibited by viral proteins and RNAs through their interactions, as well as by the viral infection-mediated upregulation of certain miRNAs. Here, we review the antiviral functions of TRIM25 and highlight its significance regarding innate immunity, particularly in antiviral defense and viral immune evasion.
... 15 As the majority of current proximity-inducing small molecules for E3s recruit substrates to the physiological substrate binding component (e.g. the substrate adaptor of Cullin RING E3s), 11 we focused our efforts towards liganding the PRYSPRY substrate binding domain of TRIM25. TRIM25 has been reported to ubiquitinate a number of different substrates, possibly in some cases mediated through RNA binding, 16 including RIG-I, 15,17,18 DDX3X 19 and ZAP, [20][21][22] with diverse roles in immune regulation, cancer signalling pathways and antiviral activity. [23][24][25][26] As such, TRIM25 is a promising candidate for redirection to a variety of neosubstrates, and the development of novel chemical tools Fig. S1A, ESI; † (E) k obs (h −1 ) plotted against concentration (mM), fitted using a straight line fit. ...
The tripartite motif (TRIM) family of RING-type E3 ligases catalyses the formation of many different types of ubiquitin chains, and as such, plays important roles in diverse cellular functions, ranging from immune regulation to cancer signalling pathways. Few ligands have been discovered for TRIM E3 ligases, and these E3s are under-represented in the rapidly expanding field of induced proximity. Here we present the identification of a novel covalent ligand for the PRYSPRY substrate binding domain of TRIM25. We employ covalent fragment screening coupled with high-throughput chemistry direct-to-biology optimisation to efficiently elaborate covalent fragment hits. We demonstrate that our optimised ligand enhances the in vitro auto-ubiquitination activity of TRIM25 and engages TRIM25 in live cells. We also present the X-ray crystal structure of TRIM25 PRYSPRY in complex with this covalent ligand. Finally, we incorporate our optimised ligand into heterobifunctional proximity-inducing compounds and demonstrate the in vitro targeted ubiquitination of a neosubstrate by TRIM25.
... Like other TRIM proteins, TRIM25 encodes a central coiled-coil domain that drives the formation of obligate antiparallel dimers, and an N-terminal RING domain, which catalyzes autoubiquitination and ubiquitination of other target proteins (Fig. 6a) 25 . The RING domain is also an additional site of dimerization 24 , and TRIM25, therefore, likely forms higher-order multimers. ...
ZAP is an antiviral protein that binds to and depletes viral RNA, which is often distinguished from vertebrate host RNA by its elevated CpG content. Two ZAP cofactors, TRIM25 and KHNYN, have activities that are poorly understood. Here, we show that functional interactions between ZAP, TRIM25 and KHNYN involve multiple domains of each protein, and that the ability of TRIM25 to multimerize via its RING domain augments ZAP activity and specificity. We show that KHNYN is an active nuclease that acts in a partly redundant manner with its homolog N4BP1. The ZAP N-terminal RNA binding domain constitutes a minimal core that is essential for antiviral complex activity, and we present a crystal structure of this domain that reveals contacts with the functionally required KHNYN C-terminal domain. These contacts are remote from the ZAP CpG binding site and would not interfere with RNA binding. Based on our dissection of ZAP, TRIM25 and KHNYN functional anatomy, we could design artificial chimeric antiviral proteins that reconstitute the antiviral function of the intact authentic proteins, but in the absence of protein domains that are otherwise required for activity. Together, these results suggest a model for the RNA recognition and action of ZAP-containing antiviral protein complexes.
... However, whether the mutation in ZnF2 impacts its pocket structure, or even the folding of the protein, remains unknown. Interestingly, the mutations within ZnF2 (C88R) and ZnF4 (H191R), but not those in the CpG-binding pocket, enhanced the inhibitory effect of ZAP on JEV translation [46]. At the same time, these mutations increased the association between ZAP and TRIM25, an E3 ubiquitin ligase that has been identified as a cofactor of ZAP [11,12,46]. ...
... Interestingly, the mutations within ZnF2 (C88R) and ZnF4 (H191R), but not those in the CpG-binding pocket, enhanced the inhibitory effect of ZAP on JEV translation [46]. At the same time, these mutations increased the association between ZAP and TRIM25, an E3 ubiquitin ligase that has been identified as a cofactor of ZAP [11,12,46]. This suggests that the integrity of ZAP's zinc-fingers and their interactions with RNA can alter how ZAP interacts with co-factor to attenuate translation of specific viral RNAs. ...
... ZAP inhibits HIV-1 mRNA translation by competitively binding with eIF4A and thus interferes with the interaction between eIF4G and eIF4A [86] ( Figure 3C). The interaction between ZAP and TRIM25 showed a significant positive correlation with JEV translation inhibition [46], however the exact role of TRIM25 in facilitating translation inhibition of ZAP remains unclear. ...
The zinc-finger antiviral protein (ZAP) is a restriction factor that proficiently impedes the replication of a variety of RNA and DNA viruses. In recent years, the affinity of ZAP's zinc-fingers for single-stranded RNA (ssRNA) rich in CpG dinucleotides was uncovered. High frequencies of CpGs in RNA may suggest a non-self origin, which underscores the importance of ZAP as a potential cellular sensor of (viral) RNA. Upon binding viral RNA, ZAP recruits cellular cofactors to orchestrate a finely tuned antiviral response that limits virus replication via distinct mechanisms. These include promoting degradation of viral RNA, inhibiting RNA translation, and synergizing with other immune pathways. Depending on the viral species and experimental set-up, different isoforms and cellular cofactors have been reported to be dominant in shaping the ZAP-mediated antiviral response. Here we review how ZAP differentially affects viral replication depending on distinct interactions with RNA, cellular cofactors, and viral proteins to discuss how these interactions shape the antiviral mechanisms that have thus far been reported for ZAP. Importantly, we zoom in on the unknown aspects of ZAP's antiviral system and its therapeutic potential to be employed in vaccine design.
... The human ZAP protein, which primarily targets CpG dinucleotides in viral RNA sequences, demonstrates the ability to impair the infection of various negative-and positive-sense single-stranded RNA viruses (116,125,126). Acting as a post-transcrip tional RNA restriction factor within target cells, ZAP effectively targets viruses like filoviruses, coronaviruses, retroviruses, and alphaviruses (51,116,127,128). ...
The coronavirus disease 2019 (COVID-19) pandemic remains an international health problem caused by the recent emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of May 2024, SARS-CoV-2 has caused more than 775 million cases and over 7 million deaths globally. Despite current vaccination programs, infections are still rapidly increasing, mainly due to the appearance and spread of new variants, variations in immunization rates, and limitations of current vaccines in preventing transmission. This underscores the need for pan-variant antivirals and treatments. The interferon (IFN) system is a critical element of the innate immune response and serves as a frontline defense against viruses. It induces a generalized antiviral state by transiently upregulating hundreds of IFN-stimulated genes (ISGs). To gain a deeper comprehension of the innate immune response to SARS-CoV-2, its connection to COVID-19 pathogenesis, and the potential therapeutic implications, this review provides a detailed overview of fundamental aspects of the diverse ISGs identified for their antiviral properties against SARS-CoV-2. It emphasizes the importance of these proteins in controlling viral replication and spread. Furthermore, we explore methodological approaches for the identification of ISGs and conduct a comparative analysis with other viruses. Deciphering the roles of ISGs and their interactions with viral pathogens can help identify novel targets for antiviral therapies and enhance our preparedness to confront current and future viral threats.
... This partnership between TRIM25 and ZAP is also essential for TRIM25-mediated restriction of JEV RNA translation (Figure 1) [80]. In this context, ZAP (but not TRIM25) bound viral RNA to inhibit its translation, and was potentiated by the interaction with TRIM25, although no mechanism was proposed ( Figure 1). ...
Flaviviruses comprise a large number of arthropod-borne viruses, some of which are associated with life-threatening diseases. Flavivirus infections are rising worldwide, mainly due to the proliferation and geographical expansion of their vectors. The main human pathogens are mosquito-borne flaviviruses, including dengue virus, Zika virus, and West Nile virus, but tick-borne flaviviruses are also emerging. As with any viral infection, the body’s first line of defense against flavivirus infections is the innate immune defense, of which type I interferon is the armed wing. This cytokine exerts its antiviral activity by triggering the synthesis of hundreds of interferon-induced genes (ISGs), whose products can prevent infection. Among the ISGs that inhibit flavivirus replication, certain tripartite motif (TRIM) proteins have been identified. Although involved in other biological processes, TRIMs constitute a large family of antiviral proteins active on a wide range of viruses. Furthermore, whereas some TRIM proteins directly block viral replication, others are positive regulators of the IFN response. Therefore, viruses have developed strategies to evade or counteract TRIM proteins, and some even hijack certain TRIM proteins to their advantage. In this review, we summarize the current state of knowledge on the interactions between flaviviruses and TRIM proteins, covering both direct and indirect antiviral mechanisms.
... Notably, a large percent of them (19 out of 56) have been reported to function in regulating cellular immune and/or antiviral responses together with TRIM25, including Zinc-Finger Protein ZCCHC3 41,42 , and m6A reader IGF2BP2 43 (Fig. 4c). Remarkably, a comparison of the TRIM25 WT poly(I:C) vs. untreated proximal interactomes revealed a similar pattern, including ZCCHC3, IGF2BP2, and Zinc-Finger Antiviral Protein ZC3HAV1 (also known as ZAP [44][45][46][47][48] (Fig. 4d). These data indicate that TRIM25 and G3BP1 may co-condensate to facilitate the antiviral functions and innate immune responses upon viral invasion. ...
... IGF2BP2 showed significant co-localization with both G3BP1 and TRIM25 ΔPTFG foci, consistent with its association with both proteins (Fig. 4e). Unlike them, ZC3HAV1 co-localized with TRIM25 ΔPTFG foci, but not with poly(I:C)-induced SGs, likely reflecting a tight interaction between ZC3HAV1 and TRIM25 [44][45][46][47][48] (Fig. 4e). We also examined the subcellular localization of AVPs with GFP-TRIM25 condensates in G3BP dKO HeLa cells. ...
Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25’s ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.
... 24,26,28,29 Antiviral activity requires TRIM25 and PARP13 co-localization at membranes and the RNA-binding domains of both proteins. 11,17,18,24,28,30,31 Ablation of TRIM25's putative RNA-binding domain in its SPRY domain decreases PARP13 interaction, 28 suggesting the interaction may be mediated via protein-RNA interaction and indicating the possibility of co-regulation of shared targets. ...
... Both proteins require their RNA-binding domains to perform their roles in the innate antiviral response. 17,18,30,31 To interrogate whether the requirement of RNA binding for these proteins is related to their interaction with one another, we used CLIP-seq data to identify shared targets of TRIM25 and PARP13. Although no datasets of TRIM25 are published for 293T cells, comparison with CLIP-seq data from HeLa cells transiently expressing T7-tagged TRIM25 28 revealed that 87% of all identified PARP13 targets were also bound by TRIM25, which is far greater than could be expected by chance ( Figure 5A, Fisher's exact test, p = 2.16 3 10 À126 ). ...
... To assess the degree to which PARP13 RNA binding contributes to its well-documented association with TRIM25, 24,26,28,29,31 we performed co-immunoprecipitation experiments. We transfected GFP-tagged PARP13.1, ...
The RNA-binding protein PARP13 is a primary factor in the innate antiviral response, which suppresses translation and drives decay of bound viral and host RNA. PARP13 interacts with many proteins encoded by interferon-stimulated genes (ISG) to activate antiviral pathways including co-translational addition of ISG15, or ISGylation. We performed enhanced crosslinking immunoprecipitation (eCLIP) and RNA-seq in human cells to investigate PARP13’s role in transcriptome regulation for both basal and antiviral states. We find that the antiviral response shifts PARP13 target localization, but not its binding preferences, and that PARP13 supports the expression of ISGylation-related genes, including PARP13’s cofactor, TRIM25. PARP13 associates with TRIM25 via RNA-protein interactions, and we elucidate a transcriptome-wide periodicity of PARP13 binding around TRIM25. Taken together, our study implicates PARP13 in creating and maintaining a cellular environment poised for an antiviral response through limiting PARP13 translation, regulating access to distinct mRNA pools, and elevating ISGylation machinery expression.
... ZAP binding requires not only the proper spacing of CpGs, but also, the nucleotide composition surrounding CpGs influences the ZAP's affinity for target RNAs [86,90]. In alphaviruses, the ZAP blocked the translation initiation by preventing the assembly of the cap binding complex (eIF4F) on the incoming viral gmRNAs at early times of infection ( Figure 3) [84,91]. However, since the ZAP preferentially binds to internal regions of the SINV gmRNA, it is not clear how ZAP binding prevents mRNA recruitment by the eIF4F complex, which occurs distally [80]. ...
... For alphaviruses, the longer ZAP isoforms showed a better antiviral effect [82]. A full ZAP activity requires cofactors such as the ubiquitin E3 ligase tripartite motif-containing protein (TRIM25) that also interacts with viral mRNAs [91,93]. ...
Alphaviruses can replicate in arthropods and in many vertebrate species including humankind, but only in vertebrate cells do infections with these viruses result in a strong inhibition of host translation and transcription. Translation shutoff by alphaviruses is a multifactorial process that involves both host- and virus-induced mechanisms, and some of them are not completely understood. Alphavirus genomes contain cis-acting elements (RNA structures and dinucleotide composition) and encode protein activities that promote the translational and transcriptional resistance to type I IFN-induced antiviral effectors. Among them, IFIT1, ZAP and PKR have played a relevant role in alphavirus evolution, since they have promoted the emergence of multiple viral evasion mechanisms at the translational level. In this review, we will discuss how the adaptations of alphaviruses to vertebrate hosts likely involved the acquisition of new features in viral mRNAs and proteins to overcome the effect of type I IFN.
... Blanchard et al. [13] also showed that messenger RNA (mRNA)-encoded Cas13a was effective against influenza A and SARS-CoV-2 viruses in mice and hamsters, respectively. In addition, RNA-binding proteins (RBPs), which are upregulated in virus-infected cells, have been shown to play crucial roles in suppressing or influencing host-virus interaction [14], notably zinc finger antiviral protein (ZAP) [15,16] and TRIM25 [17], and these proteins could be repurposed to achieve targeted viral inhibition. ...
Advances in synthetic biology have led to the design of biological parts that can be assembled in different ways to perform specific functions. For example, genetic circuits can be designed to execute specific therapeutic functions, including gene therapy or targeted detection and the destruction of invading viruses. Viral infections are difficult to manage through drug treatment. Due to their high mutation rates and their ability to hijack the host’s ribosomes to make viral proteins, very few therapeutic options are available. One approach to addressing this problem is to disrupt the process of converting viral RNA into proteins, thereby disrupting the mechanism for assembling new viral particles that could infect other cells. This can be done by ensuring precise control over the abundance of viral RNA (vRNA) inside host cells by designing biological circuits to target vRNA for degradation. RNA-binding proteins (RBPs) have become important biological devices in regulating RNA processing. Incorporating naturally upregulated RBPs into a gene circuit could be advantageous because such a circuit could mimic the natural pathway for RNA degradation. This review highlights the process of viral RNA degradation and different approaches to designing genetic circuits. We also provide a customizable template for designing genetic circuits that utilize RBPs as transcription activators for viral RNA degradation, with the overall goal of taking advantage of the natural functions of RBPs in host cells to activate targeted viral RNA degradation.
