The complex interplay between encephalomyocarditis virus and the host defence system

ABSTRACT

A variety of animals can be infected by encephalomyocarditis virus (EMCV). EMCV is the established causative agent of myocarditis and encephalitis in some animals. EMCV causes high fatality in suckling and weaning piglets, making pigs the most susceptible domestic animal species. Importantly, EMCV has zoonotic potential to infect the human population. The ability of the pathogen to avoid and undermine the initial defence mechanism of the host contributes to its virulence and pathogenicity. A large body of literature highlights the intricate strategies employed by EMCV to escape the innate immune machinery to suit its “pathogenic needs.” Here, we also provide examples on how EMCV interacts with certain host proteins to dampen the infection process. Hence, this concise review aims to summarize these findings in a compendium of decades of research on this exciting yet underappreciated topic.

Introduction

EMCV is a Cardiovirus genus member of the Picornaviridae family, which is a non-enveloped, single-stranded RNA virus. The genetic makeup of this virus is similar to that of the foot-and-mouth disease virus (FMDV) [1]. EMCV has the potential to induce myocarditis and encephalitis in different mammals, leading to the development of neurological ailments, reproductive abnormalities, and diabetes [2,3]. Both clinical symptoms and mortality rates differ markedly, depending on the host species [4]. Neurological manifestations, associated with myocarditis in pigs have been documented globally, causing significant financial damage to the global swine sector [5,6]. Global distribution of EMCV has been confirmed through aetiological and serological studies, supporting previous theories suggesting that the majority of human cases are likely to be asymptomatic or go unnoticed [7,8]. Importantly, EMCV has a strong zoonotic potential for transmission to humans via a faecal-oral transmission route [7,8].

The genome of EMCV viral RNA is composed of a 5‘-untranslated region (5-UTR), a single open reading frame (ORF), and a 3’-UTR. Figure 1 shows that the single ORF codes for four structural proteins (VP4, VP2, VP3, and VP1) and eight non-structural proteins (L^pro, 2A, 2B, 2C, 3A, 3B, 3C, and 3D) [9,10]. The viral replication complex is primarily composed of non-structural proteins, while EMCV structural proteins are crucial for viral entry, uncoating, and assembly. Pathogen recognition receptors (PRRs) are host receptors that recognize conserved molecular structures known as pathogen-associated molecular patterns (PAMPs) primarily by Toll-like receptors (TLRs), RIG-I (retinoic acid-inducible-gene-I)-like receptors (RLRs), Nod-like receptors (NLRs), and C-type lectin receptors (CLRs) [11,12]. Upon detection of pathogens, different immune responses against microorganisms are promptly activated through the stimulation of inflammatory cytokines, chemokines, and type I interferons (IFNs). Indeed, RNA viruses can be sensed by these host PRRs [11–13].

Figure 1.

The viral genome structure of Encephalomyocarditis virus (EMCV). The viral genome contains a 5′-untranslated region (5′UTR), a large open reading frame (ORF) including the L, VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C, and 3D coding regions, and a 3′UTR.

These host responses, which have a crucial and significant impact on the outcome of the infection, can be triggered by viruses. The key aspect is when the host develops a state that restricts the replication and transmission of the virus, known as an antiviral status. The activation of signalling pathways that lead to the activation of type I IFNs is commonly accomplished [14]. After being released, the IFNs attach to IFN receptors (IFNAR1 and/or IFNAR2) to initiate downstream Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathways, ultimately resulting in the extensive expression of a range of IFN-stimulated genes (ISGs). These outcomes lead to a wide range of cellular effects, such as protection against viruses, inhibition of cell growth, and enhancement of the ability of the host to adapt to infections [15].

To meet its “infective” requirements, EMCV has developed different tactics to combat immune responses. This review primarily examines the mechanisms employed by EMCV to evade the immune system, which in turn regulates host responses. We will also provide evidence on how EMCV interacts with certain host proteins to its own detriment. Finally, we discuss the consequences of these strategies on both the virus and the host.

It is important to disclose foreseeable limitations to what has been reported so far on the topic of EMCV interplay with the host. Viruses are highly variable in nature. This goes with EMCV and the study of EMCV pathogenesis in particular cell types. It is clear that the plethora EMCV studies reported to date utilize particular strains to study host pathogen interaction biology and we should be cognizant that some findings may not be representative of every EMCV variant in nature. Therefore, even if studies reveal the immune escape mechanism of a particular strain on a particular cell type, this mechanism may not be applicable to other variants. However, it is important to emphasize that all EMCV strains are similar in antigenicity and that only minor evolutionary changes of capsid structures have taken place over time in the population [16]. Indeed, a detailed phylogenetic analysis revealed the limited genetic variability of EMCV [17]. In saying this, it is important to disclose that EMCV may exhibit different immune escape properties depending on the infected cell type. As a result, the results of studies on one cell type may not reflect that in other cell types. The reliance on in vitro methods, such as cell culture systems and molecular biology techniques, has been crucial in advancing our understanding of EMCV patho-biology. However, it is important to recognize that these methods have their limitations and may not fully recapitulate the complexity of viral infections in natural hosts. Taking all of these factors into account, it is reasonable to suggest that the EMCV interplay with the host innate immune machinery, which are evolutionarily conserved, could occur in different cell types that are equipped with such “antiviral weaponry.”

Future research in this field will likely benefit from the integration of multiple methodologies, including advanced in vitro co-culture 3D models, novel imaging techniques, and alternative host in vivo approaches. This multimodal approach may help us better understand the mechanisms underlying EMCV infections, identify novel targets for therapeutic interventions, and ultimately contribute to the control and prevention of infectious diseases.

EMCV proteins regulate host innate immune responses

Efficient detection of viral RNAs by host sensors is necessary for a successful immune response against viruses [14]. In the interaction between EMCV and the host, the involvement of various downstream signalling mediators, including nuclear factor kappa B (NF-κB) and IFN regulatory factors (IRFs), is crucial [18–21]. These mediators induce pro-inflammatory and antiviral IFN responses, respectively. To undermine identification, viruses typically encode proteins that either conceal viral RNAs from detection, or directly attach to and hinder (or split) immune receptors or other molecules engaged in antiviral signalling pathways.

Inhibiting IFN production

The structure, receptor distribution, and tissue-specific biological activities of IFNs is variable. It is crucial to note that all IFNs induce an “antiviral state” in the host [22]. Type I IFNs are an essential group of antiviral agents that can directly or indirectly regulate viral replication and spread. Certainly, engagement of PRRs by recognizing PAMPs has the potential to trigger the activation of type I IFNs [23]. Nevertheless, viruses have developed tactics to counter the signalling cascades of the IFNs system to guarantee their survival and spread. EMCV, in fact, inhibited the production of IFN through the activity of its proteins 3C, 2C, VP2 and 3A (as shown in Figure 2). 3C can disrupt the assembly of the TANK-TBK1-IKKε-IRF3 complex, which is associated with the TRAF family member and NF-κB activator, thereby blocking type I IFN signalling [18]. Additionally, it can also suppress the pro-inflammatory NF-κB signalling pathway mediated by TRAF6 [19]. 2C interacts with the dsRNA-sensing RLR protein, melanoma differentiation-associated gene 5 (MDA5), to inhibit downstream IFN-β signalling [20]. VP2 breaks down the RLR sensors MDA5 and mitochondrial antiviral signalling protein (MAVS), as well as their downstream adaptor protein TBK1, using both the proteasome and lysosomal pathways [21]. EMCV 3A also inhibited IFN signalling pathway. Interestingly, 3A does not act directly on the signalling pathway adaptor protein, but rather degrades the host chaperone protein heat shock protein 27 (HSP27), which in turn influences the stability of MDA5 for the purpose of evading the innate immune response to promote self-proliferation [24].

Figure 2.

Inhibition of IFN production by encephalomyocarditis virus (EMCV). When EMCV infects host cells with caveolin-mediated endocytosis, pattern recognition receptor MDA5 recognizes viral RNA and activates a series of innate immune response pathways. EMCV proteins VP2, 2C, 3C and 3A are shown in orange circles, cellular proteins DDX56, HSP27, HSP90β, DHX29, TRIM13, RAVER1 and ZFYVE1 are shown in green ellipses. EMCV 3C interferes with the formation of the TANK-TBK1-IKKε-IRF3 complex to inhibit type I interferon (IFN) signalling and suppress the pro-inflammatory TRAF6-mediated NF-κB signalling pathway. 2C can interact with MDA5 and inhibit the IFN-β signalling pathway. VP2 degrades RLR sensors MDA5 and MAVS, thereby inhibiting IFN-β production. 3A also inhibited IFN signalling pathway. Interestingly, 3A does not act directly on the signalling pathway adaptor protein, but rather degrades the host’s chaperone protein heat shock protein 27 (HSP27), which in turn influences the stability of MDA5 for the purpose of evading the innate immune response to promote self-proliferation. In addition to the immune escape function of the proteins of the virus itself, EMCV can hijack some of the host proteins to inhibit IFN production, thus facilitating its own proliferation. Cellular protein DDX56 competitively binds nuclear transport protein KPNA3 and KPNA4 with IRF3, thereby inhibiting IRF3 nuclear translocation and IFN-β production. HSP27 serves as a molecular chaperone, positively regulates EMCV-triggered RLR/MDA5 signal pathway by stabilizing the expression of MDA5 and viral protein 3A can degrade the expression of HSP27. HSP90β targets MAVS to inhibit IFN pathway activation. TRIM13 is an E3 ubiquitin ligase, it is a negative regulator of MDA5-mediated IFN-β production. DHX29 identifies dsRNA and selectively associates with MDA5 to strengthen antiviral defence. RAVER1 as a specific MDA5-interacting protein, it was associated with MDA5 upon viral infection. ZFYVE1, a zinc-finger FYVE domain-containing protein, is a negative regulator of MDA5, and lack of ZFYVE1 promotes MDA5 expression, thereby effectively promoting antiviral responses.

Regulating inflammatory responses

The innate immune system, as the first line of defence against pathogens, utilizes PRRs to detect PAMPs. RNA viruses are detected by host PRRs including TLRs, RIG-I-like helicases (RLHs), and NLR family, pyrin domain-containing 3 (NLRP3) [11–13]. EMCV infection can activate inflammatory signalling pathway to enhance its replication and further propagation in host cells [25].

In response to viral infections, the activation of pro-inflammatory cytokines IL-1β and IL-18 is significantly influenced by the NLRP3 [26]. After being activated, NLRP3 creates a protein compound called the NLRP3 inflammasome by combining with an apoptosis-associated speck-like protein that contains a caspase recruitment domain (ASC) and pro-caspase-1. As a result, caspase-1 undergoes autocatalytic cleavage, transforming it into its active state. This enables the proteolytic processing of pro-IL-1β and pro-IL-18 into their biologically active forms downstream [27].

Figure 3 illustrates the significant contribution of the inflammatory reaction caused by EMCV to its pathogenesis. The viroporins of Picornavirus 2B are recognized as transmembrane pore-forming viral proteins that modify the permeability of membranes to ions through the creation of membrane channels. These proteins also play a role in various viral functions [25]. The activation of the NLRP3 inflammasome has been demonstrated due to EMCV viral protein 2B. EMCV increases the local Ca²⁺ concentration in the cytoplasm by stimulating Ca²⁺ flux from intracellular storages through the action of its viroporin 2B, thereby activating the NLRP3 inflammasome [28].

Figure 3.

Regulation of inflammatory responses by encephalomyocarditis virus (EMCV). EMCV protein is shown in orange circle. EMCV 2B, acts as viroporins protein, can increase the local Ca²⁺ concentration in the cytoplasm by stimulating Ca²⁺ flux, thereby activating the NLRP3 inflammasome.

Modulation of apoptosis

Apoptosis, also known as programmed cell death, occurs in multicellular organisms. Crucially, apoptosis serves as a vital innate defence mechanism for the host to hinder viral replication and eradicate cells infected by viruses. As a result, viruses have developed tactics to hinder or postpone apoptosis while replicating, guaranteeing the survival of cells until an adequate number of new viruses are generated [29,30]. Certainly, the prevention of cell death is crucial for the virulence of EMCV. In vivo, Schwarz et al. showed that the inhibition of apoptosis by NF-κB is crucial for the pathogenesis of EMCV [31]. Mice were rescued from EMCV-induced clinical disease by eliminating the p50 subunit of NF-κB, which was associated with reduced viral titers in cardiac tissues. This implies that the inhibition of apoptosis during EMCV infection necessitates the involvement of NF-κB signalling.

EMCV 2A is an important virulence factor [3]. Crucially, the 2A protein has demonstrated its ability to hinder programmed cell death. According to the report, BHK-21 cells infected with EMCV lacking 2A resulted in the activation of caspase 3, leading to cell apoptosis [32] (Figure 4). This indicates that the presence of the 2A protein is necessary to prevent apoptosis. Additionally, the EMCV 2A protein was found to hinder apoptosis by interacting with annexin A2 via the JNK/c-Jun pathway [33].

Figure 4.

Modulation of apoptosis and autophagy by encephalomyocarditis virus (EMCV). EMCV proteins are shown in orange circles. 2A dampens apoptosis through caspase 3 activation, interacts with activated caspase-3 to inhibit apoptosis. 2A also can elevate NF-κB activity to increase inflammatory genes expression. EMCV nonstructural protein 3A and capsid protein VP1 colocalized with LC3. 2C and 3D are also involved in EMCV-induced autophagy by activating ER stress molecules and regulating the proteins expression associated with UPR pathway. SIDT2 is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation.

Regulating the processes of autophagy

Autophagy, a degradation pathway reliant on lysosomes, not only contributes significantly to cellular balance but also actively engages in diverse physiological and pathological mechanisms. Autophagy, which is a crucial element of the host’s defence system against various microorganisms such as bacteria, viruses, and protozoa [34], plays a significant role in protecting the body. The acidic environment in autolysosomes gives rise to the activation of enzymes essential for degrading pathogens that have invaded the host cell.

The relationship between EMCV replication and autophagy has been extensively documented (Figure 4). EMCV may potentially manipulate autophagosomes in order to create a conducive environment for viral infection. The presence of EMCV infection leads to an augmentation in the quantity of vesicles with both double and single membranes in the cytoplasm of host cells. These vesicles are considered as a characteristic feature of autophagic structures within cells, and the replication of EMCV is necessary for the initiation of autophagy [35]. The non-structural protein 3A and VP1 capsid protein of EMCV are found in the same location as microtubule-associated protein 1 light chain 3 (LC3), which is the mammalian equivalent of yeast Atg8, a significant protein involved in autophagy and serves as a marker. Furthermore, besides LC3, the protein sequestosome 1 (SQSTM1, also known as p62), which experiences enhanced degradation during autophagy, is commonly employed to evaluate autophagic flux. In their study, Zhang et al. [35], demonstrated that LC3-I/-II conversion and degradation of p62/SQSTM1 were triggered by EMCV infection. The involvement of EMCV nonstructural proteins 2C and 3D in EMCV-induced autophagy has also been demonstrated. This occurs through the activation of endoplasmic reticulum (ER) stress molecules and the regulation of protein expression related to the unfolded protein response pathway [36]. In short, evidence to date shows that the initiation of autophagy is critical for EMCV as it hijacks this machinery to create a conducive environment to favour its own growth and propagation.

Host proteins involved in modulating EMCV-mediated innate immune responses

The constant interaction between viruses and host cells results in specific relationships with different viral species, which to some extent limits the host range of viruses and facilitates their infection. On the other hand, it is also an opportunity for cells to induce certain antiviral responses. Gaining knowledge about the interplay of EMCV and host cells will enhance our comprehension of the virus infection mechanism and aid in devising appropriate strategies for prevention and treatment. Importantly, EMCV benefits from this by evading its detection by the host to favour its propagation and spread to other neighbouring cells [21]. Here, we provide examples on how certain host proteins can promote and others that dampen EMCV infection.

DExD/H RNA helicases

RNA helicases have significant functions in every aspect of RNA metabolism, encompassing transcription, translation, processing, and degradation. ATP is utilized by these extremely conserved enzymes to attach, separate, and eliminate RNA structures and RNA-protein complexes [37].

Inhibiting EMCV infection

DHX29 functions as a dsRNA co-detector and specifically binds to MDA5 to boost RNA identification when exposed to high molecular weight polyinosinic-polycytidylic acid [HMW-Poly (I:C)] or infection with an RNA virus. It is important to note that RNA helicases have significant functions in various biological processes, such as the initiation of protein translation and the formation of stress granules [37]. Furthermore, apart from their potential participation in the recognition of innate immunity and antiviral defence [38], numerous RNA helicases have been recognized as crucial elements in the initiation of protein translation [39]. In one study, the function of DHX29 in MDA5-triggered EMCV-specific type I IFN signalling was shown, shedding light on how DHX29 identifies dsRNA and selectively associates with MDA5 to strengthen antiviral defence [40]. This study also offered valuable information on the molecular process involved. DHX29 interacts with MDA5 upon engagement, leading to downstream signalling of type I IFN.

Promoting EMCV infection

In EMCV infection, DDX56, a different RNA helicase, also has a significant function [41]. Enhanced expression of DDX56 facilitated the replication of EMCV, while its absence hindered EMCV replication. As a result, the expression of type I IFN was enhanced during EMCV infection following the knockdown of DDX56. In an activation cascade of MDA5 signalling triggered by EMCV, DDX56 hinders the phosphorylation of IFN regulatory factor 3 (IRF3) and prevents its translocation to the nucleus, ultimately resulting in the inhibition of type I IFN production [41].

Heat shock proteins (HSPs)

Heat shock protein proteins (HSPs) are molecules that promote the folding of other proteins. The majority of HSPs are triggered by stress, as they have a crucial role in protecting cells that are subjected to challenging circumstances [42]. HSPs can be located within cells and are released, especially when encountering stress. So HSPs are often regarded as biomarkers of danger. Hence, the immune system reacts to existing unfavourable cellular circumstances. Significantly, HSPs are linked to both inflammatory and anti-inflammatory reactions [43].

Inhibiting EMCV infection

Many viral proteins interact with HSPs. During EMCV infections, HSP27 functions as a host factor that combats EMCV infection and enhances the generation of type I IFNs triggered by EMCV [24]. Furthermore, HSP27 acts as a molecular chaperone, enhancing the EMCV-induced MDA5 signalling pathway through the stabilization of MDA5 expression.

Promoting EMCV infection

HSP90β is another HSP involved in EMCV infection. Li et al. [44] discovered that HSP90β has a beneficial impact on the replication of EMCV. The inhibition of herpes simplex virus replication is achieved through the formation of a complex between HSP90β and TBK1 [45]. Additional investigations demonstrated that HSP90β engages with TBK1 in the context of EMCV infection, in conjunction with MAVS and IRF3. To summarize, HSP90β hinders the type I IFN reactions in the RLRs pathway activated by EMCV by focusing on the regulation of crucial adapter molecules, namely MAVS, TBK1, and IRF3 [46].

Tripartite motif (TRIM) proteins

TRIM proteins, which belong to the tripartite motif family, act as ubiquitin E3 ligases and play diverse roles in multiple cellular processes. TRIM proteins are crucial in protecting the host from viral infections [46]. Certain TRIM proteins directly counteract specific stages in the viral life cycle, while others control signalling pathways triggered by innate immune receptors, thus adjusting antiviral cytokine responses. Furthermore, TRIM proteins play a role in virus-triggered autophagy and autophagy-dependent elimination of viruses [47]. Considering the significant function of TRIM proteins in antiviral limitation, it is not unexpected that numerous viruses have developed successful tactics to counteract the antiviral impacts of particular TRIM proteins.

Inhibiting EMCV infection

TRIM proteins can attach to ubiquitin linkers, which are responsible for determining the destiny of altered target proteins [47]. The essential role of TRIM22 E3 ubiquitin ligase activity in mediating antiviral activity against EMCV has been demonstrated [48]. Specifically, TRIM22 interacts with EMCV 3C protease (3C^pro) and mediates its ubiquitination.

Promoting EMCV infection

On the other hand, TRIM56 poses no antiviral threat to EMCV, but it does to other RNA viruses, indicating the adaptability and selectivity of TRIM56 in combating specific RNA viruses [49]. TRIM13 is another E3 ubiquitin ligase. It is not only a negative regulator of MDA5-mediated IFN-β production but also may positively regulate RIG-I function [50].

Other host proteins

Other host proteins can modulate EMCV host immune antiviral responses. RAVER1, a ribonucleoprotein PTB-binding protein 1, specifically interacts with MDA5. Crucially, MDA5 is linked to RAVER1 during EMCV infection [51]. The transcription of type I IFN genes induced by EMCV infection in vitro is significantly suppressed by genetic knockdown of RAVER1, indicating that RAVER1 is essential for the MDA5-mediated activation of downstream antiviral genes. Another important host protein that modulates EMCV host responses is SIDT2. SIDT2 moves internalized extracellular double-stranded RNA from the endocytic compartments to the cytoplasm to activate the immune system [52]. The impaired production of antiviral cytokines in EMCV-infected mice lacking SIDT2 indicates that SIDT2 plays a crucial role in regulating host antiviral responses [52]. MDA5 was found to be negatively regulated by Guanidine-binding protein (GBP) and ZFYVE1, a protein containing a zinc-finger FYVE domain [53]. The findings of this research demonstrated that Zfyve1^-/- mice exhibited a notable resistance to lethality caused by EMCV, indicating that ZFYVE1 functions as a distinct inhibitor of MDA5-mediated innate immune reactions.

Future perspectives

Despite the host’s development of robust natural defence mechanisms, primarily facilitated by type I IFNs, to combat viral infections, EMCV has developed tactics to exploit host immune responses for its own survival. EMCV employs a range of viral proteins to limit different aspects of the IFN response network through degradation or disruption of their functions. EMCV employs various strategies to hinder the IFN system, which is expected to prevent the antiviral impact of IFN, enabling EMCV to effectively establish its infection.

This review provides an overview of the tactics employed by EMCV to elude the host’s natural defence system, encompassing disruption of the IFN signalling pathway, modulation of inflammatory reactions, manipulation of autophagy, and control of apoptosis. We also highlighted how host cellular proteins participate in defending against EMCV infections. Numerous gaps in knowledge still need to be explored and investigated, particularly concerning the regulation of the virus in diverse hosts throughout various stages of infection. Furthermore, the validation of numerous observations due to the excessive expression of viral proteins in different cellular systems is yet to be verified in vivo.

To gain a deeper understanding of the immune evasion mechanism employed by EMCV, it is necessary to conduct additional comparative studies in both human and animal cells. These studies can offer valuable references for comprehending host-viral interactions within the context of EMCV-mediated immune evasion. Conducting comparative research could assist in the identification of crucial molecules and pathways that are targeted by EMCV. Furthermore, it is necessary to gain a deeper comprehension of the intricate molecular processes that take place during EMCV infection, encompassing innate antiviral immune reactions, apoptosis, autophagy, and inflammatory responses.

The development of disease after EMCV infection may be determined by a delicate equilibrium between cytokines that have antiviral effects and those that have harmful effects, which are linked to cytokine-induced inflammatory responses. For example, EMCV attachment to C-C motif chemokine receptor 5 (CCR5) in macrophages rapidly induces inflammatory cytokine production [54]. CCR5-induced inflammation plays a role in controlling the replication and proliferation of EMCV [55]. This further reinforces the idea that the production of pro-inflammatory cytokines has a positive impact on the virulence of EMCV. Hence, it is of utmost significance to identify crucial host elements implicated in the development of EMCV. Our expectation is that this evaluation will improve our comprehension of virus-host interactions while EMCV infection occurs and offer valuable perspectives for creating effective vaccines or antiviral substances targeting EMCV. Given that EMCV is a neurotrophic virus, it is becoming increasingly important to understand how EMCV maintains its infectious nature in the brain. As with other neurotrophic viruses like Japanese encephalitis virus (JEV) and Zika virus (ZEV) [56], the way EMCV evades the immune responses in such sites may be similar to other neurotrophic viruses.

Funding Statement

This work was supported by the National Natural Science Foundation of China [32260037], Talent Introduction Research Projects of Northwest Minzu University [No. xbmuyjrc2023015] and Fundamental Research Funds for the Central Universities [31920230160 and 31920230162].

Author contributions

JX drafted and wrote the manuscript. RF designed the artwork. AI and RF have revised the manuscript and provided valuable suggestions. All authors have read and approved the final manuscript.

Data availability statement

Data sharing not applicable – no new data generated.

Disclosure statement

No potential conflict of interest was reported by the author(s).