The DEAD-box protein family is a conserved family of RNA helicases containing a DEAD-box domain, named after the conserved Asp-Glu-Ala-Asp (DEAD) sequence (Pathak et al., 2007). DDX21 mediates innate immunity and regulates the viral replication of viruses such as foot-and-mouth disease virus (FMDV) (Abdullah et al., 2021), dengue virus (DENV) (Dong et al., 2016) and Borna disease virus (BDV) (Watanabe et al., 2009). Furthermore, DDX21 has been shown to inhibit SVV replication, but its effects can be weakened by SVV 3C^pro, which induces caspase-dependent degradation of DDX21 and suppresses host antiviral immunity, limiting the cell antiviral response (Zhao et al., 2022). DHX30, a DEAD family member, contributes to the biosynthesis of mitochondrial ribosomes. Zinc-finger antiviral protein (ZAP) recruits DHX30 to unfold and degrade viral RNA (Ye et al., 2010). Although DHX30 inhibits SVV replication through its helicase activity, SVV 3C^pro can mediate the cleavage of DHX30 helicase at the Q220 site, leading to loss of its ability to inhibit SVV replication (Wen et al., 2022).

hnRNPs are a widely functional family of RNA-binding proteins mainly located in the cell nucleus (Jean-Philippe et al., 2013), hnRNP A1 can bind to viral proteins and modulate the replication of viruses such as the nucleocapsid proteins of porcine epidemic diarrhoea virus (PEDV) (Li et al., 2018). In addition, hnRNP A1 can interact directly with viral RNA sequences, including the coding region of the human papillomavirus 16 (HPV16) E7 sequence (Zheng et al., 2020). Research has shown that the protease activity of SVV 3C^pro mediates the degradation and translocation of hnRNP A1, which enhances the replication of SVV (Song et al., 2021). Similarly, hnRNP K participates in virus replication through interaction with the 5′ UTR of enterovirus 71 (EV71) (Lin et al., 2008) and FMDV (Liu et al., 2020). Knockdown of hnRNP K significantly inhibits SVV replication, whereas hnRNP K overexpression promotes virus proliferation. These studies suggest that intracellular hnRNP K contributes to SVV replication. SVV infection leads to the cleavage, degradation, and cytoplasmic redistribution of hnRNP K due to the activity of 3C^pro. 3C^pro induces the degradation of hnRNP K through the caspase pathway and cleaves hnRNP K at Q364. The cleaved fragment hnRNP K (365–464) promotes virus replication, whereas full-length hnRNP K exerts a similar effect. In contrast, the noncleaved fragment hnRNP K (1–364) tends to inhibit virus replication (Song et al., 2022b).

Nucleolin (NCL) is involved in several cellular processes, such as RNA transcription, ribosome formation, nuclear-cytoplasmic transport, and posttranscriptional regulation of mRNA (Becherel et al., 2006; Durut and Sáez-Vásquez, 2015). Moreover, NCL is linked with viral proliferation and plays a crucial role in virus replication through nucleocytoplasmic redistribution (Greco et al., 2012; Han et al., 2021). An increase in the NCL expression levels and its cleavage are induced by SVV, which drives NCL redistribution outside the cell nucleus. The 3C^pro protein of SVV relocates NCL to the cytoplasm and cleaves it at Q545. Cleaved NCL facilitates virus replication, and the proteolytic activity of the 3C^pro protein regulates the cleavage and relocalization of NCL (Song et al., 2022c).

Cytoplasmic poly (A)-binding protein 1 (PABPC1) interacts with eukaryotic translation initiation factor 4G (eIF4G) and promotes the binding of the 60S ribosomal subunit to the 48S preinitiation complex in the final step of initiation, facilitating translation initiation (Tahiri-Alaoui et al., 2014; Smith et al., 2017). SVV replication is inhibited by PABPC1; however, SVV 3C^pro cleaves PABPC1 at Q437, which disrupts protein synthesis and interferes with host immunity, thereby promoting virus replication (Xue et al., 2020).

SVV 3C^pro inhibits RNA metabolism regulation, gene expression, and the immune response mediated by the aforementioned factors, thereby evading host defence mechanisms and providing a conducive environment for its replication.

The innate immune response represents the crucial initial defence against pathogenic invasion in the host organism. RIG-I-like receptors (RLRs), including retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), detect viral RNA and trigger the activation of mitochondrial antiviral signalling protein (Yoneyama et al., 2005; Paludan and Bowie, 2013). Upon activation, MAVS undergoes dimerization on the mitochondrial membrane, where it subsequently associates with TNF receptor-associated factor 3 (TRAF3) (Yoneyama et al., 2005; Tang and Wang, 2009). TRAF3 then recruits TRAF-related NF-κB-activating kinase (TANK), and TANK transduces upstream signals to TANK-binding kinase 1 (TBK1). TBK1 induces the phosphorylation of interferon regulatory factor 3/7 (IRF-3/7) and nuclear transcription factor kappa B (NF-κB), resulting in their dimerization and nuclear translocation (Pomerantz and Baltimore, 1999; Taniguchi et al., 2001; Shu et al., 2013). Ubiquitination is a crucial regulatory element in this process. For RIG-I to bind to RNA and become active, TRIM25 must undergo K63 ubiquitination. TRAF3 is K63-ubiquitinated, leading to the stimulation of IRF-3 phosphorylation (Castanier et al., 2012; Zhu et al., 2020). TIR domain containing adaptor molecule 1 (TRIF) responds to dsRNA and LPS, resulting in the activation of NF-κB (Schwandner et al., 1999; Alexopoulou et al., 2001).

The 3C^pro of SVV extensively inhibits the type I interferon (IFN) pathway by impairing vital factors. SVV 3C^pro inhibits MAVS-mediated downstream signal transduction by degrading RIG-I, which is responsible for recognizing viral RNA. Second, by cleaving MAVS (Qian et al., 2017) at Q148, SVV 3C^pro interrupts its interaction with RIG-I (Wen et al., 2019), leading to reduced activation of downstream pathways. Recent studies have shown that SVV infection suppresses the interaction between MAVS and RIG-I by promoting lactate production through glycolysis, thereby facilitating virus replication (Li et al., 2023). SVV 3C^pro also possesses deubiquitinase activity, which inhibits the ubiquitination of RIG-I, TBK1, and TRAF3 (Xue et al., 2018b). In addition, SVV 3C^pro cleaves TRIF at Q159 (Qian et al., 2017). The N-and C-terminal domains of TRIF play different roles in the regulation of innate immunity and cell apoptosis. The N-terminal domain of TRIF is essential for the initiation of IFN promoter activity, whereas both the N-and C-terminal domains are involved in NF-κB activation. Despite the N-terminal domain separating from TRIF, the C-terminal domain still plays a role in initiating interferon signals, activating NF-κB, and inducing cell apoptosis. SVV 3C^pro interacts with IRF3 and IRF7, which inhibits their phosphorylation and leads to their degradation, ultimately affecting the regulation of IFN and other inflammatory genes (Xue et al., 2018a). In addition, SVV 3C^pro cleaves TANK at the E272 and Q291 sites (Qian et al., 2017), affecting its regulation of TBK1/IKKε and IRF3/7, and resulting in the inhibition of IFN production. Moreover, SVV 3C^pro mediates the cleavage of NF-κB by caspase 3 (Fernandes et al., 2019), suppressing its regulation of cytokine expression. SVV 3C^pro utilizes multiple mechanisms to deregulate crucial factors in the IFN pathway and thus widely inhibits the production and transfer of IFN, which enables it to evade the host’s immune response (Figure 1).

Stress granules (SGs) are cytoplasmic structures composed of RNA and RNA-binding proteins that form in response to various stressors, including hypoxia, environmental toxins, and viral infections (Wen et al., 2020). The formation of SGs induced by viral infection inhibits the synthesis of viral proteins, exerting antiviral effects prior to the upregulation of host antiviral protein transcription. During infection, the double-stranded RNA (dsRNA) formed by the virus activates PKR, leading to eIF2α phosphorylation and subsequent promotion of SGs formation (McEwen et al., 2005; Yamasaki and Anderson, 2008). The RNA-binding protein GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) binds to mRNA and aggregates, forming the core of SGs (Yamasaki and Anderson, 2008). Eukaryotic translation initiation factor 4G 1 (eIF4G1) is a key regulatory protein involved in translation initiation. G3BP1 can bind to and sequester eIF4G1, promoting stress-induced SGs formation and playing a role in mRNA transport and stability (Kedersha et al., 2016). Picornavirus 2A or L protein inhibits SGs formation by interfering with the eIF4GI-G3BP interaction (Yang et al., 2019). SVV infection induces the formation of stress granules (SGs) during the early stages of infection, but these SGs dissipate as the infection progresses to the late stages. Additionally, SVV infection transiently induces PKR and eIF2α phosphorylation-dependent SGs formation. SVV replication plays a crucial role in SGs formation. The cleavage of eIF4G1 by SVV. 3C^pro disrupts the eIF4GI-G3BP1 interaction, thus impairing SGs formation and contributing to enhanced viral replication (Wen et al., 2020).

The NLRP3 inflammasome, which is a large multiprotein complex that senses danger signals inside and outside the cell and initiates an inflammatory response, mainly consists of three components: NLRP3 (NOD-like receptor protein 3), ASC (apoptotic speck-like protein containing a caspase recruitment domain), and caspase-1. Upon the appearance of danger signals, such as pathogen infection, inflammatory stimulation or cell damage, the NLRP3 inflammasome can be activated, and once activated, the inflammasome can activate inflammatory cytokines such as IL-1β, IL-18 and gasdermin D (GSDMD) by stimulating caspase-1 activation. When activated, GSDMD forms pores and triggers pyroptosis, which is characterized by the rupture of the cell membrane and the release of intracellular contents. GSDMD belongs to the gasdermin family, which is a group of proteins with functions in cell membrane perforation and induction of inflammation. GSDMD has a self-inhibitory structure located between the N-and C-termini. When cleaved by proteases such as caspase-1, the N-terminus of GSDMD (GSDMD-N) is released and inserted into the cell membrane, forming pores. This process consequently results in the exchange of intracellular and extracellular substances, which ultimately leads to pyroptosis (Shi et al., 2015; Choudhury et al., 2022).

SVV 3C^pro cleaves NLRP3 at Q305, resulting in the inhibition of NLRP3 inflammasome formation. Concurrently, the protease directly cleaves pig GSDMD at Q193 and Q277, leading to release of the N-terminus of GSDMD. The cleavage of GSDMD 1–275 by caspase-1 triggers pyroptosis. Additionally, because GSDMD Q277 is proximal to the caspase-1-mediated cleavage site, GSDMD 1–277 may induce pyroptosis (Wen et al., 2021a). Research has shown that after infection with SVV markedly increases pig GSDMD cleavage and cell pyroptosis, confirming the inducible effect of SVV 3C^pro on GSDMD cleavage and pyroptosis. This finding suggests that SVV 3C^pro can independently trigger the inflammatory response of the NLRP3 inflammasome (Figure 2).

Apoptosis is an essential programmed cell death mechanism involved in physiological and pathological processes, such as development, growth, immunity, and disease. Apoptosis is a widely recognized defence mechanism of the host and serves to ensure the death of cells that have been infected by viruses. There are two types of pathways that facilitate cell apoptosis, extrinsic and intrinsic, and these pathways are executed by the caspase family of cysteine proteases (Cohen, 1997; Elmore, 2007). The extrinsic pathway is initiated by external triggers and is regulated by membrane death receptors such as CD95 (Fas/APO-1) and tumour necrosis factor receptor 1 (TNFR1). When apoptosis is induced, caspase-8 is activated and triggers downstream effector molecules, including caspase-1, caspase-3, caspase-6, and caspase-7 (Muzio et al., 1996). The intrinsic pathway involves the activation of mitochondrial-related proteins. When host cells are subjected to factors such as infection and injury, the mitochondrial apoptosis pathway is stimulated, resulting in changes in mitochondrial membrane permeability and the release of various apoptotic factors, including cytochrome C (Galluzzi et al., 2012). This process leads to the activation of caspase-9 and downstream caspase-3, ultimately leading to cell apoptosis.

At the early stages of SVA infection, the apoptotic pathway is not activated, which may allow the virus to complete its replication cycle before cell death/lysis and at the later stages of infection, the induction of cell apoptosis by SVV may function as a mechanism for facilitating the release and/or dissemination of the virus from infected cells (Kaminskyy and Zhivotovsky, 2010; Sun et al., 2016; Fernandes et al., 2019). The ability of 3C^pro to induce apoptosis in cells seems to remain consistent in small RNA viruses, including enterovirus 71 and poliovirus. Studies suggest that the SVV 3C^pro protein stimulates caspase-3, caspase-8, and caspase-9 and indicate that this protein invokes cell apoptosis through both mitochondrial and exogenous death receptor pathways (Liu et al., 2019). Although SVV 3C^pro induces cell apoptosis through its protease activity, it does not directly cleave PARP 1, which is a hallmark of cell apoptosis (Oliver et al., 1998; Liu et al., 2019). Emerging studies have shown that NF-κB likely plays a vital role in safeguarding host cells from small nuclear RNA virus-induced apoptosis (Oliver et al., 1998; Schwarz et al., 1998). A few studies have postulated that caspase 3-mediated cleavage of NF-κB encourages cell apoptosis (Kang et al., 2001; Kim et al., 2005), whereas SVV 3C^pro might impede innate immunity while promoting cell apoptosis by mediating the cleavage of caspase 3 through NF-κB (Fernandes et al., 2019; Figure 3). Notably, infection with human immunodeficiency virus (HIV) and African swine fever virus (ASFV) induces caspase-mediated NF-κB-p65 cleavage, thereby enhancing viral replication or inducing cell apoptosis after completion of the viral replication cycle (Vallée et al., 2001; Coiras et al., 2008). 3C^pro has a unique structural domain that binds to native phospholipid molecules, such as cardiolipin (CL) and phosphatidylinositol-4-phosphate (PI4P). CL can activate SVA 3C^pro activity in a homologous manner, leading to the cleavage of viral polyproteins and host proteins (such as NLRP3 and MAVS), disrupting host responses and ensuring viral replication. This binding serves as a positive regulatory mechanism for 3C^pro activity and promotes viral replication (Zhao et al., 2023). The replication of most positive-sense RNA viruses necessitates remodelling of cell membranes, converting them into virus replication organelles (ROs). The lipid microenvironment within ROs, which are rich in PI4P, is crucial for the replication of enterovirus RNA (Belov and van Kuppeveld, 2012). PV 3C^pro exhibits a wide and specific PIP-binding affinity for phospholipids, including PI4P. The binding of SVA 3C^pro to PI4P is hypothesized to induce membrane transformation and facilitate viral genome replication (Belov and van Kuppeveld, 2012; Zhao et al., 2023).

Autophagy is a cellular degradation process that breaks down proteins, organelles, and other substances into small molecules for cellular metabolism. During autophagy, the cell forms and releases autophagosomes containing degraded substances (Klionsky and Codogno, 2013). Modulating the mTOR pathway may affect the virus-induced autophagy response (Zhu et al., 2012). Autophagy enhancement can result from inhibiting AKT activation, whereas AKT activation can decrease autophagy (Hay and Sonenberg, 2004). AMP-activated protein kinase (AMPK) modulates autophagy by inhibiting the mTOR pathway. Phosphorylation of the mTOR binding partner Raptor, which is controlled by AMPK, is necessary to inhibit the mTOR pathway (Yang and Klionsky, 2010). The MAPK signalling pathway, which is essential in the signal transduction network of eukaryotes, plays a vital role in vital processes such as cell proliferation, differentiation, autophagy, apoptosis and the stress response (Gwinn et al., 2008).

A previous study revealed that SVV infection induces autophagy via the PKR-like ER protein kinase (PERK) and the activating transcription factor 6 (ATF6) signalling pathways associated with endoplasmic reticulum stress. Autophagy promotes SVV infection in pig cells, and the research results further demonstrate the involvement of the PERK and ATF6 pathways in autophagy induction. Decreasing the expression of PERK or ATF6 can inhibit SVV replication (Hou et al., 2019). However, in cultured human cells, SVV triggers an autophagic response that inhibits viral replication (Wen et al., 2021b). Research has demonstrated that VP1, VP3, and 3C^pro can cooperatively activate the AKT-AMPK-MAPK-mTOR signalling pathway during SVV infection in host cells, leading to the induction of cellular autophagy. The expression of 3C^pro upregulates the levels of p-ERK1/2 MAPK, p-p38 MAPK, and p-AKT and induces no significant change in the p-AMPK levels. The synergistic effect of p-ERK1/2 MAPK, p-p38 MAPK, and p-AKT causes a reduction in the p-MTOR levels. The transfection of cells with 3C^pro alone has no significant effect on the LC3-II level. However, transfection with SVV VP1 or 3D alone results in a significant alteration in the LC3-II level. VP1 expression promotes the phosphorylation of AKT and AMPK, leading to a reduction in the p-MTOR levels. Moreover, VP3 can activate the ERK1/2 MAPK-MTOR and p38 MAPK-MTOR pathways to facilitate autophagy. Therefore, the findings suggest that viral proteins can work synergistically to initiate autophagy, which in turn enhances viral replication (Song et al., 2022a). Currently, no studies have been investigated the impact of 3C^pro on the PERK and ATF6 UPR signalling pathways.

The receptor protein SQSTM1/p62 plays a significant role in the process of selective autophagy by recruiting ubiquitinated proteins or pathogens to autophagosomes for degradation (Bjørkøy et al., 2005; Lamark et al., 2017). Previous research reports have shown that SQSTM1 targets ubiquitin-dependent and ubiquitin-independent viral capsids for autophagic clearance, including FMDV, chikungunya virus (CHIKV) (Berryman et al., 2012; Judith et al., 2013). By targeting the SVV VP1 and VP3 proteins to autophagosomes, SQSTM1 suppresses viral replication. Research has revealed that SVV 3C^pro proactively cleaves SQSTM1/p62 at E355, Q392, and Q395, thereby producing an SQSTM1 cleavage product that cannot effectively induce selective autophagy or inhibit SVV replication (Wen et al., 2021b; Figure 4).