The subcellular location of viral replication largely defines the site of Z-NAs production. This spatial context typically influences whether ZBP1 sensing occurs in the nucleus or the cytoplasm (Ingram et al., 2019). Using IAV as a model, recent work illustrates how this spatial distinction drives distinct cell death modalities. IAV recruits ZBP1 to the nucleus via viral ribonucleoproteins (vRNPs) (Figure 1A). Here, nuclear sensing triggers “nuclear necroptosis” characterized by nuclear envelope rupture prior to plasma membrane lysis (Figure 1B) (Zhang et al., 2020). Crucially, this nuclear breakdown drives a hyper-inflammatory profile by releasing nuclear DAMPs (e.g., IL-33, HMGB1) (Gautam et al., 2024). This unique topography is immunologically decisive. In sharp contrast, poxviruses like Mpox virus and vaccinia virus (VACV) replicate entirely in the cytoplasm (Park and Walsh, 2022). In these cytoplasmic contexts, ZBP1 activation triggers canonical necroptosis primarily targeting the plasma membrane without the distinct nuclear envelope breakdown observed in IAV (Figures 1C, D). Even within the nucleus, outcomes vary. While IAV drives nuclear rupture, other nuclear viruses like herpesviruses typically lack this phenotype (Guo et al., 2015). However, the precise reason for this absence remains unclear. The presence of antagonists like ICP6 (Shanmugam et al., 2021), which intercept signaling, makes it difficult to determine whether these viruses naturally lack the capacity for nuclear rupture or if the phenotype is simply masked by viral inhibition. Consequently, comparing these viral classes underscores a critical insight that ZBP1 sensing is a plastic mechanism, governed by the specific interplay between viral localization and immune evasion strategies.

The Z-NAs that activate ZBP1 arise from multiple sources that include viral replication byproducts and virus-induced abnormalities in host transcription. Defective viral genomes (DVGs), truncated species, and palindromic sequences can fold into local double-stranded RNA and adopt the Z conformation, as observed in infections with IAV and Reovirus (Guo et al., 2018; DeAntoneo et al., 2022; He et al., 2025). Nevertheless, these species do not fully explain ZBP1 activation in all contexts, since robust ZBP1 activation is observed even during influenza infection with a low burden of DVGs. Building on this, recent work proposes a model termed Disruption of Transcriptional Termination (DoTT), primarily characterized during IAV infection, where viral interference with host RNA processing machinery generates large numbers of host transcripts with excessively long 3’ extensions (Figure 1) (Yin et al., 2025). These extensions are enriched for endogenous retroelements, specifically SINEs and LINEs. Their inverted repeats promote intramolecular pairing to form stable dsRNA that has the potential to adopt Z conformations, which in turn represents a potential source of ZBP1 ligands (Nacken et al., 2021; Yin et al., 2025). Taken together, these observations suggest that ZBP1 may sense not only products of active, high-intensity viral replication but also virus-driven damage to the host transcriptional machinery, positioning it as a sensitive indicator of pathological transcriptional stress in infected cells. At the same time, detecting Z-NAs in situ remains challenging because the Z conformation is intrinsically labile and can be shielded by viral or host proteins, potentially leading to underestimation of its true abundance (Yin et al., 2023). Future studies should develop more sensitive enrichment, sequencing, and spatial mapping strategies to resolve the spatiotemporal dynamics of Z-NAs during infection with greater precision.

Viral replication dynamics suggest fluctuating Z-RNA abundance across infection stages (Mishra et al., 2024). While direct quantification remains challenging, biophysical and structural evidence may involve a threshold-like behavior. At the molecular level, ZBP1 activation depends on higher-order assembly (Peng et al., 2022). Viral Z-NAs recognition by its N-terminal Zα domains triggers a conformational shift exposing the RIP homotypic interaction motif (RHIM) (Xie et al., 2024), enabling homotypic interactions with RIPK3 and RIPK1 (Koerner et al., 2024). This contact drives the rapid oligomerization of these proteins into amyloid-like signaling complexes (Polonio et al., 2025). Within this dense architecture, the signaling components are brought into close physical proximity. This organization may facilitate proximity-driven kinase activation and help overcome inhibitory constraints (Koerner et al., 2024; Amusan et al., 2025). Specifically, recent studies identify an inhibitory splice isoform (ZBP1-S) lacking RHIM domains as a key modulator of this threshold. ZBP1-S competitively binds Z-NAs and constrains oligomerization of the full-length isoform (ZBP1-L), thereby modulating the ligand threshold required for robust pathway activation (Figure 1) (Cai et al., 2024; Nagata et al., 2024). Thus, this RHIM-dependent core functions as a central signaling hub to orchestrate convergent PANoptosis and inflammatory cascades. Given the mechanistic complexity and viral diversity, we distinguish established evidence from emerging conceptual models in Table 1.

A fundamental component of the antiviral strategies driven by ZBP1 is the induction of PANoptosis (Malireddi et al., 2023). This integrated cell-death paradigm eliminates infected host cells to halt viral spread (Figure 2). Conceptually, the essence of this integrated process lies in its parallel, multi-arm architecture, which ensures that even when viruses evolve inhibitors against a single pathway, the host can still clear infection through alternative routes. Mechanistically, ZBP1 engages its RHIM domain to recruit and activate RIPK3, driving necroptosis via MLKL phosphorylation and plasma membrane rupture (Koerner et al., 2024; Amusan et al., 2025). In parallel, the ZBP1–RIPK3 platform functions as a signaling scaffold that recruits RIPK1 and the adaptor FADD, thereby leading to activation of the initiator caspase-8. Once activated, caspase-8 not only cleaves and activates the executioner caspases-3 and -7 to trigger classical apoptosis (Rodriguez et al., 2022), but also acts as a critical node of cross-talk that promotes assembly of the NLRP3 inflammasome, resulting in caspase-1 activation (Malireddi et al., 2023). Consequently, caspase-1 performs dual effector functions, including the cleavage of gasdermin D (GSDMD) to generate membrane pores and drive pyroptosis, and it processes pro-IL-1β and pro-IL-18 into their mature, secreted forms. This multi-pathway design provides substantial defense redundancy (Evavold et al., 2021; Dong et al., 2024). Therefore, even in the face of numerous viral caspase inhibitors and other immune-evasion proteins, the pathway ensures convergence on cell death, such that at least one downstream pathway remains functionally active. Ultimately, this strategy represents an advanced host countermeasure to viral immune evasion, sacrificing individual infected cells to prevent organism-wide dissemination.

Beyond its role in cell death, ZBP1 orchestrates a robust, cell-death-independent immune response composed of two key arms. First, it rapidly induces NF-κB dependent inflammation (Li et al., 2024a). Second, it cooperates with the cGAS-STING pathway to amplify type I interferon (IFN-I) production (Gomes et al., 2024). Together, these coordinated actions create a powerful antiviral state (Figure 2). Mechanistically, ZBP1 efficiently engages NF-κB signaling through its RHIM domain. By recruiting RIPK1 to assemble a signaling platform that activates the IKK complex, resulting in IκBα degradation and consequent nuclear translocation of NF-κB. Once in the nucleus, NF-κB promptly induces a wide array of pro-inflammatory genes, including TNF-α and IL-6, creating a local inflammatory milieu that functions as an immediate immune response at sites of infection (Imai et al., 2024; Koerner et al., 2024). Concurrently, functional synergy between ZBP1 and the cGAS-STING axis serves as a principal amplifier. This is initiated when ZBP1-driven necroptosis promotes the release of mitochondrial and other endogenous DNA into the cytosol, which is then sensed by cGAS to activate STING. This activation, occurring via the TBK1-IRF3 axis, robustly increases IFN-I production. Subsequently, the secreted IFN-I functions synergistically with pro-inflammatory cytokines, such as TNF-α and IL-6, produced by the NF-κB pathway (Yang et al., 2021; Ding et al., 2025). Thus, by functioning as an upstream apical regulator, ZBP1 tightly integrates rapid, localized inflammation with a potent systemic interferon response, promoting a comprehensive antiviral state essential for the efficient clearance of viral replication.

A fundamental mechanism limiting ligand generation (Strategy 1) involves the utilization of replication strategies that intrinsically minimize Z-NAs formation, thereby reducing the likelihood of detection by the ZBP1 surveillance system(Figure 3). The formation of Z-NAs is closely associated with imperfect replication processes, such as defective viral genomes (DVGs) produced by low-fidelity RNA polymerases or complementary RNA strands arising from overlapping transcriptional units (Brennan and Sun, 2024). Accordingly, any viral mechanism that enhances replication fidelity or reduces dsRNA by-products may indirectly facilitate evasion of ZBP1 (Gribble et al., 2021; Dupre and Volmer, 2023). Although there is currently no direct evidence that selection pressure from ZBP1 has driven the evolution of polymerase fidelity, certain virus-encoded proteins do have the potential to diminish immunostimulatory RNA. For instance, coronaviruses ubiquitously encode conserved enzymes that minimize immunostimulatory byproducts. Specifically, the exonuclease nsp14 confers proofreading capacity to reduce defective viral genomes (Ogando et al., 2020), while the endoribonuclease nsp15 cleaves viral RNA intermediates (Zhang et al., 2023a). Together, these enzymatic activities limit the accumulation of immunostimulatory RNA during replication (Gribble et al., 2021; Otter et al., 2024), thereby reducing the potential for Z-NA formation and subsequent detection by ZBP1 (Yang et al., 2025b). While these mechanisms likely evolved primarily for genomic stability rather than specifically to evade ZBP1, they objectively reduce the likelihood of ZBP1 activation. Furthermore, a computational study hypothesized that SARS-CoV-2 nsp13 might convert Z-RNA to A-RNA through its helicase activity, although this remains experimentally unvalidated, thereby directly erasing the ligand for ZBP1 (Herbert and Poptsova, 2022). Notably, future experimental validation of nsp13’s potential Z-RNA disruption could reveal novel strategies of immune avoidance that physically destabilize PAMP molecules, potentially offering a new perspective on how viruses modulate host innate immunity.

However, when rapid viral replication makes the generation of Z-NAs unavoidable, viruses must deploy a distinct evasion strategy (Strategy 2) to sequester these ligands (Figure 3). Some viruses, particularly poxviruses, have evolved a “signal-masking” strategy whereby, during the early phase of viral replication, proteins bearing a Zα domain are expressed to competitively bind and conceal newly produced viral Z-NAs (Koehler et al., 2021). Classic examples include Mpox virus (Li et al., 2023b) and VACV (Hsu et al., 2024), which both encode the multifunctional E3 protein containing an N-terminal Zα domain that structurally mimics host ZBP1 to competitively bind Z-NAs (Park et al., 2020). Crucially, as an early-gene product expressed shortly after viral entry, E3 acts as a pre-emptive shield that sequesters Z-NAs immediately upon their generation, thereby blocking ZBP1 recognition at the source (Soday et al., 2019; Pham et al., 2023). While intact E3 effectively suppresses ZBP1-dependent necroptosis by preventing ligand access (Zhang et al., 2023b), its mutation or deletion triggers robust cell death, resulting in impaired replication and markedly reduced virulence (Wang et al., 2024). This creates the necessary conditions for subsequent genome replication, late-stage protein synthesis, and the assembly of progeny virions. Therefore, the Zα domain of E3 is a key determinant of successful poxviral replication, achieving effective shielding from host immune surveillance by directly competing with ZBP1 for the ligand at the molecular level.

Distinct from ligand sequestration, herpesviruses employ a strategy (Strategy 3) that targets downstream signaling rather than the ligand itself (Figure 3). Members of the subfamily Alphaherpesvirinae exemplify this strategy. For example, similar to the well-characterized HSV-1, pseudorabies virus (PRV) (Lyu et al., 2020) and equine herpesvirus 1 (EHV-1) (Vereecke et al., 2021) each encode viral RHIM-containing proteins (vRHIMs) homologous to ICP6. These early-gene products function as molecular mimics, interacting with the RHIM domains of ZBP1 or RIPK3 through homotypic binding (Shanmugam et al., 2021; Lodha et al., 2023). Consequently, such interactions exert a competitively inhibitory effect that disrupts proper assembly of the host ZBP1-RIPK3 signaling complex, preventing effective activation of downstream MLKL and thus blocking execution of necroptosis (Petrie et al., 2019). Similarly, a related mechanism has been identified in varicella-zoster virus (VZV), whose ORF20 protein also contains a RHIM domain and has been shown to inhibit ZBP1-dependent necroptosis (Steain et al., 2020; Verdonck et al., 2022). The evolutionary advantage of this approach likely stems from the multifunctional nature of vRHIMs, which integrate immune evasion functions into essential viral proteins (Ng et al., 2022). In this way, through molecular mimicry, herpesviruses specifically disrupt the integrity of the ZBP1 signaling axis. This ensures that even upon ZBP1 activation, host cells fail to execute cell death, thereby preserving a stable intracellular environment for viral genome replication and virion maturation (Wu et al., 2023).

For viruses employing antagonists to evade surveillance, re-activating ZBP1 represents a rational strategy to abort replication and restore host defense (Figure 4). Because direct ZBP1 agonists are currently unavailable, most translational strategies rely on repurposing existing drugs to activate the pathway indirectly. One widely used upstream approach is IFN-I therapy. As ZBP1 is an interferon-stimulated gene, IFN-α/β treatment markedly increases its expression (Yang et al., 2020), thereby lowering the threshold required for ZBP1 activation during viral infection, including settings where viral proteins antagonize upstream sensing (Koehler et al., 2017; Oh and Lee, 2023). Another strategy focuses on increasing endogenous ligands that can activate ZBP1. DNA hypomethylating agents, such as 5-azacytidine, can reactivate endogenous retroviruses and lead to the accumulation of cytosolic dsRNA (Noronha et al., 2024). In addition, the curaxin CBL0137 promotes the formation of Z-conformation DNA from chromatin (Wang et al., 2021; Li et al., 2024b). These nucleic acid structures can then be detected by ZBP1. Sensitization of downstream signaling has also been explored using SMAC mimetics, including birinapant. These compounds induce the degradation of cIAP1/2, thereby removing inhibitory constraints on the RIPK1–RIPK3 axis. As a result, necroptosis is more readily triggered, particularly when caspase-8 function is impaired (Ebert et al., 2015; Brumatti et al., 2016). Beyond host-directed approaches, targeting viral antagonists represents another possible direction. For example, future therapies could aim to disrupt the interaction between viral proteins such as vaccinia virus E3 and Z-NAs (Koehler et al., 2021). Although such interactions may be difficult to target structurally, interfering with viral suppression of ZBP1 could offer a potential strategy to re-engage the host’s antiviral surveillance. While ZBP1 activation strategies may benefit infections with potent viral antagonists, their utility against viruses with minimal evasion mechanisms remains uncertain, as excessive pathway engagement risks immunopathology (Li et al., 2023a; Boyd et al., 2025).

In many severe viral infections, such as severe COVID-19 or influenza, ZBP1 hyperactivation is a primary driver of lethal immunopathology and cytokine storms (Liang et al., 2024). Therefore, modulating ZBP1 signaling to avoid “auto-destructive” immune injury is a key and data-supported clinical goal. One clinically approved strategy targets ZBP1 expression (Figure 4). ZBP1 is a well-established Interferon-Stimulated Gene (ISG) (Yang et al., 2020). In severe COVID-19, ZBP1-driven inflammatory cell death creates a pathological feedback loop (Li et al., 2023a). The JAK1/JAK2 inhibitor Baricitinib, approved for severe COVID-19, is pertinent to this pathway. Its mechanism involves inhibiting JAK/STAT signaling, thereby suppressing the interferon-driven expression of ISGs, including ZBP1 (Kalil et al., 2021). A second strategy, which has advanced into human clinical trials, targets the downstream kinase RIPK1. Advanced, brain-penetrant RIPK1 inhibitors such as DNL104 and SAR443060 (DNL747) have completed Phase I and Phase Ib clinical trials for human neuroinflammatory diseases, including ALS and Alzheimer’s (Vissers et al., 2022). Finally, specific evidence demonstrates that the downstream MLKL-executioner arm of necroptosis can be pharmacologically targeted in viral infection models. The selective RIPK3 inhibitor UH15–38 effectively blocks IAV-induced necroptosis by inhibiting RIPK3 activation and subsequent MLKL phosphorylation (Gautam et al., 2024). Furthermore, in vivo studies with Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) demonstrated that pharmacological inhibition of MLKL decreased fatality (Li et al., 2025). The aforementioned strategies are indirect, focusing on upstream expression or downstream effectors. In contrast, direct targeting of ZBP1 has been difficult, mainly because the protein does not contain clear druggable binding pockets. More recently, covalent PROTACs have been developed to selectively degrade ZBP1 (Huang et al., 2025). These bifunctional molecules recruit an E3 ubiquitin ligase to ZBP1, leading to its ubiquitination and proteasomal degradation. Collectively, the integration of established upstream suppression, downstream blockade, and emerging direct degradation strategies provides a comprehensive framework for clinical intervention. This multi-level approach allows for the precise management of ZBP1-driven immunopathology.