The Strategy of Picornavirus Evading Host Antiviral Responses: Non-structural Proteins Suppress the Production of IFNs

Viral infections trigger the innate immune system to produce interferons (IFNs), which play important role in host antiviral responses. Co-evolution of viruses with their hosts has favored development of various strategies to evade the effects of IFNs, enabling viruses to survive inside host cells. One such strategy involves inhibition of IFN signaling pathways by non-structural proteins. In this review, we provide a brief overview of host signaling pathways inducing IFN production and their suppression by picornavirus non-structural proteins. Using this strategy, picornaviruses can evade the host immune response and replicate inside host cells.

Viruses develop various strategies to inhibit secretion of IFNs and promote viral replication inside host cells. Mounting evidence shows that infecting viruses can evade IFN response either by suppressing IFN production or by blocking IFN induction of interferon-stimulated gene factors (ISGs) (Zinzula and Tramontano, 2013;Fensterl et al., 2015). Viral non-structural proteases play an important role in this process. In this review, we summarize our current knowledge of the role of picornavirus non-structural proteases in antagonizing IFN induction via different signaling pathways to inhibit host antiviral responses.

SIGNALING PATHWAYS INDUCING IFN PRODUCTION
When viruses infect organisms, the host innate immune system detects the presence of pathogen-associated molecular patterns via host pattern recognition receptors (PRRs) (Vaccari et al., 2014;Coccia and Battistini, 2015). These include transmembrane PRRs such as Toll-like receptors (TLRs), cytosolic RIG-like RNA helicases such as melanoma differentiation-associated gene (MDA-5), retinoic acid induced gene-I (RIG-I), and other molecules (Barbé et al., 2014;Wu and Chen, 2014). PRRs recruit a number of specific adaptor proteins to trigger a downstream signaling cascade and activate three major pathways to produce IFNs: the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (Coccia and Battistini, 2015), the mitogenactivated protein kinase (MAPK), and the IFN regulatory factor (IRF) pathways (Akira et al., 2006;Honda and Taniguchi, 2006). IFNs can signal in an autocrine or paracrine manner to induce hundreds of ISGs that fortify host defenses (Figure 2; Pham et al., 2016).
Induced IFNs exert their activity by binding and activating IFN receptors. Type I IFN receptors are composed of interferonα/β receptors 1 and 2 subunits (IFNAR1 and IFNAR2) (Olière et al., 2011). Type III IFNs receptors are composed of two different chains, the IFN-λR1 high-affinity chain and IL-10R2 low-affinity chain (Chasset and Arnaud, 2017). The binding of type I IFNs with its receptor leads to the phosphorylation of Janus kinase1 (JAK1) and tyrosine kinase 2 (TYK2). These activated kinases then phosphorylate STAT1 and STAT2 and promote STAT1 and STAT2 heterodimerization. The resulting heterodimer interacts with IRF9 to form a heterotrimeric complex called interferon-stimulated gene factor 3 (ISGF3) (Peng et al., 2017). This complex then translocate to the nucleus and binds a 12-15 bp IFN-α/β-stimulated response element (ISRE' 5 -G/ANGAAAN2GAAACT-3 ), which regulates the transcription of over 300 IFN-stimulated genes, some of which lead to IFN-α and IFN-β expression (Figure 2). Type III IFNs have almost the same process with that of type I IFNs (Blaszczyk et al., 2016;Chasset and Arnaud, 2017).
Type II IFN receptor is composed of two subunits, IFNGR1 and IFNGR2. After the binding of IFN-γ proteins to IFNGR, STAT1 homodimers is formed and bind to IFN-γ-activated site (GAS) enhancer elements with the help of the promoters of IFN-stimulated genes resulting in the production of genes encoding pro-inflammatory cytokines and apoptotic factors. Furthermore, IFN-γ can also activate STAT3 homodimers, which result in the production of pro-inflammatory cytokines and anti-inflammatory cytokines IL-10 (Gonzáleznavajas et al., 2012).
Although type III and type I IFNs share many similarities in their promoter regions and binding sites for transcription factors (Onoguchi et al., 2007;Segundo et al., 2011), for instance, IFNβ and IFN-λ1/2/3 promoters contain IRF-and NF-kβ-binding sites; IFN-α promoters have IRF-binding sites (Iversen and Paludan, 2010;Durbin et al., 2013;Reid and Charleston, 2014), the expression of type III and type I IFNs is regulated through different mechanisms (Numasaki, 2009). For example, type III FIGURE 2 | The overview of picornavirus non-structural proteins suppress the production of IFNs. When viruses infect organisms, the host innate immune system detects the presence of pathogen-associated molecular patterns via host pattern recognition receptors, then recruit a number of specific adaptor proteins to trigger a signaling cascade leading to the establishment of an antiviral state based on IFN and proinflammatory cytokines induction. The green arrays represent observations that have yet to be associated with a specific mechanism of action of the production of IFNs. The scissors illustrate the inhibition of viral non-structural proteins in the production of IFNs.
interferons are produced mainly by antigen-presenting cells and epithelial cells (Chasset and Arnaud, 2017), while the major cell type responsible for type I IFN production is the pDC (Mackern-Oberti et al., 2015;Chasset and Arnaud, 2017). Type I IFNs are induced by mitochondrial-associated MAVS, whereas type III IFNs are stimulated by peroxisome-associated MAVS (Odendall et al., 2014). In addition, IRF1 plays a unique role in type III IFN induction while IRF3 and IRF7 play vital roles in type I IFN production (Österlund et al., 2007).

PICORNAVIRUS NON-STRUCTURAL PROTEINS INHIBIT IFN PRODUCTION TO COUNTERACT HOST ANTIVIRAL RESPONSES
While the host secretes IFNs to defense against viral infection, viruses have also developed effective immune evasion mechanisms to counteract the host's antiviral responses. Numerous studies have demonstrated that picornavirus proteases can cleave adaptors, receptors and regulators involved in the signaling pathways controlling IFN induction to inhibit production of type I IFNs Lei et al., 2013). Besides, picornavirus non-structural proteins play important roles in the suppression of IFNs by down-regulating host gene expression and blocking the secretory pathway (Table 1).
However, in some picornaviruses, cooperation between nonstructural proteins leads to inhibition of IFN induction (Sim et al., 2005;Chase and Semler, 2012).

Lpro of FMDV
Foot-and-mouth disease virus Lpro is a kind of papain-like cysteine protease, which was first identified by Strebel and Beck (1986). FMDV's RNA genome encodes a polyprotein, Lpro is located near its N-terminus (residue ∼2330) and there are two forms of FMDV Lpro, Laboratory (201 aa) and Lb (173 aa) (Kirchweger et al., 1994). FMDV infection selectively induces IFN-α1 mRNA, and IFN-β mRNA levels become elevated only after a significant duration of infection (24 h) (de Los Santos et al., 2006). By inhibiting IFN production (including type I and type III IFNs) at the transcriptional and translational levels, FMDV Lpro down regulates the host innate immune response to FMDV infection. FMDV Lpro can repress IFNβ transcription by reducing IFN-stimulated gene products (Li D. et al., 2016) decreasing IFN-β mRNA levels during early infection and inhibiting activation of NF-κB via degradation of the NF-κB subunit p65/RelA and ubiquitination of RIG-I, TBK1, and TRAF3/6, resulting decreased IRF-3/7 protein expression (de Los Santos et al., 2007;Wang et al., 2011). Additionally, Lpro contributes to induce the cleavage of host eukaryotic translation initiation factor 4γ (eIF4G) (Devaney et al., 1988;Belsham et al., 2000), shutting off host cap-dependent mRNA translation, thus  limiting the synthesis of host proteins (de Los Santos et al., 2006), which may possibly include type I IFNs.
Foot-and-mouth disease virus Lpro also antagonizes IFN-λ1: Lpro's catalytic activity and SAP domain are involved in the suppression of IFN-λ1 induction . In addition, by disrupting activation of NF-κB and IRFs and inhibiting IFN-λ1 expression induced by RIG-I/MDA5, FMDV Lpro inhibits IFN-λ1 promoter activation .

Lpro of Cardiovirus
Cardiovirus polyproteins begin with short N-terminal Leader (L) sequences, EMCV Lpro (∼67 residues) and TMEV Lpro (∼76 residues) contains common zinc-finger and acidic domains. Although cardiovirus Lpro is different from FMDV Lpro and does not function as a protease, it represses IFN-α/β synthesis during viral infection. In eukaryotes, nucleocytoplasmic transport of RNA and protein relies on the Ran-GTPase system. EMCV Lpro directly interacts with Ran and disrupts the RanGDP-GTP gradient leading to inhibition of nucleocytoplasmic transport (Porter et al., 2006;Ma, 2007), suppressing the production of IFN. EMCV Lpro hinge domain plays a major role in the interaction with Ran GTPase (Bacotdavis and Palmenberg, 2013). EMCV Lpro interferes with the transactivation function of IRF3 suppressing IRF3-mediated IFN-α/β production (Hato et al., 2010). Studies have demonstrated that TMEV Lpro can block the production of type I IFN at the transcriptional level ( Van et al., 2001), this transcriptional inhibition is correlated with inhibition of IRF-3 dimerization (Ricour et al., 2009).

2A of Enteroviruses
Enteroviruses 2A has protease activity (Racaniello, 2007), which can not only process the viral polyprotein (Toyoda et al., 1986), but also cleave a variety of host proteins and inhibit the host translation whose function is quite similar to Lpro in FMDV.
Enterovirus 71 2A cleaves MAVS from the outer membrane of mitochondria. The cleaved fragments are released into the cytoplasm where they effectively inactivate downstream signaling and cleave MDA5, thus preventing IRF3 phosphorylation, down regulating production of type I IFNs and increasing viral replication (as it was shown in Figure 2; Wang et al., 2013). During this process, EV71 2A cleaves at MAVS residues Gly209, Gly251, and Gly265, with a strong preference for cleavage at Gly251 . Similarly, Rhinovirus 2A inhibits the production of IFN by cleaving MAVS (Mukherjee et al., 2011). CVB3 (coxsackievirus B3) 2A and poliovirus (PV) also mediate the cleavage of MAVS and MDA5, exerting the same functions in inhibiting type I IFNs (Feng et al., 2014a).
Enterovirus 71 can still inhibit induction of downstream IFNstimulated genes, although the mechanisms of EV71 disruption of IFN signaling have been controversial. Lu et al. (2012) reported that EV71 2A acted as an IFN antagonist and that its protease activity was required for reduction of IFNAR1 levels. By reducing IFNAR1, EV71 inhibits IFN-mediated phosphorylation of Tyk2, Jak1, STAT1, and STAT2. However, Liu et al. (2014) reported that EV71 infection down regulated expression of JAK1 but did not modify expression of IFNAR1. KPNA1 is a nuclear localization signal receptor for p-STAT1. A recent study reported that EV71 infection down regulated expression of KPNA1, reducing the formation of STAT/karyopherin-α1 (KPNA1) complex and resulting reduction of the IFN .
Enterovirus 71 2A attenuates IFN-γ signaling using a mechanism that is different from type I IFNs. 2A can suppress IFN-γ signaling by reducing serine phosphorylation of STAT1 and inactivating extracellular signal-regulated kinases without affecting STAT1 expression . EV71 2A alters signal transduction of type II IFNs without affecting the protein expression of IFN-γ.
Other genera picornaviruses, such as EMCV, do not encode 2A proteinase. They are sensitive to IFN and are unable to replicate in IFN-pretreated cells. Their 2A might not have function on evading the host immune response (Morrison and Racaniello, 2009).

2B of HAV
Hepatitis A virus 2B is a peripheral membrane protein, its coding region has variants (Emerson et al., 1991(Emerson et al., , 1992(Emerson et al., , 1993, which makes it significantly larger than 2B in other picornaviruses. HAV 2B was found in close vicinity to the tubular interconnected network of mitochondrial membranes through its ability induce membrane rearrangements resulting in the influence of the production of IFNs (Gosert et al., 2000). HAV 2B suppresses MAVS signaling more effectively with the cooperation of HAV 3ABC (Ashutosh et al., 2015). HAV 2B appears to influence MAVS function without directly affecting the antigenic structure of MAVS (Paulmann et al., 2008); it also interferes with the TBK1/IKKε kinase complex. Consequently, RIG-I/MDA-5mediated activation is inhibited and inhibition of IRF-3 signaling results in efficient suppression of IFN-β synthesis (Paulmann et al., 2008).

3A of FMDV
Foot-and-mouth disease virus 3A is a partially conserved protein, it has no homologous sequence to any other known proteases, which is unique among the picornaviruses. A recent study revealed that FMDV 3A down regulates FMDV-associated IFNβ induction via FMDV 3A inhibition of RLR-mediated IFNβ induction (Li D. et al., 2016). Residues 103-153 near 3A's N-terminus interact with MDA5, RIG-I and VISA, and a 102residue region near the N-terminus mediates inhibition of the IFN-β signaling pathway (Li D. et al., 2016). FMDV 3A reduces expression of MDA5, RIG-I and VISA by decreasing their mRNA levels (Li D. et al., 2016). This finding not only reveals a novel mechanism of FMDV 3A-mediated evasion of host innate immunity but also provide a new thought to explore this kind of non-structural proteins in other picornaviruses.

3C
Picornavirus 3C is a unique cysteine protease that combines features of both serine and cysteine proteases (Di et al., 2016). Although 3C has similar spatial structures among all picornaviruses, and can inhibit IFN expression through similar pathways, including the NF-κB, Jak/STAT and IRF pathways, its specific sites of action are different.
Upon viral infection, EV71 3C can directly bind to RIG-I, impairing RIG-I's interaction with MAVS and inhibiting RIG-I-mediated type I IFN responses. It has been reported that ubiquitination of RIG-I is controlled by a tumor suppressor called CYLD (Xu et al., 2014). CYLD is a target of miR-526a, a potent IFN-β inducer, and miR-526a upregulation during viral infection is partially mediated by IRF7. By suppressing CYLD expression, miR-526a positively regulates VSV-associated type I IFN production. EV71 3C inhibits production of type I IFN by blocking miR-526a upregulation and CYLD downregulation.
CV-A16, CV-A6, and EV-D68 3C can bind to MDA5 and inhibit the interaction with MAVS, thus blocking the production of type I IFN (Rui et al., 2017). CVB3 3C also cleaves MAVS and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) at specific sites and inhibits the induction of type I IFN (Mukherjee et al., 2011).

3C of EMCV
Encephalomyocarditis virus 3C is the only cysteine protease encoded by the viral genome, and it has a high degree of substrate specificity, besides Lpro, EMCV 3C is another antagonist. TANK is an NF-κB activator, TRAF6 serves as a platform to recruit the IKK complex and kinase TAK1, and TANK negatively regulates this function (Papon et al., 2009). EMCV 3C can cleave TANK at Gln291 and Gln197 (Huang et al., 2015), disrupting formation of the TANK-TBK1-IKKε-IRF3 tetramer, decreasing TBK1-and IKKε-mediated IRF3 phosphorylation, impairing the ability of TANK to inhibit TRAF6-mediated NF-κB signaling, and reducing type I IFN production (Huang et al., 2015(Huang et al., , 2017. SG is the location for efficient interaction between viral RNA and RLRs; EMCV 3C can also block formation of SG to inhibit activation of IFN genes (Huang et al., 2017). By cleaving IRF3-5D and other key proteins, EMCV 3C inhibits JAK-STAT signaling, suppressing type I IFN production (Huang et al., 2017). EMCV 3C may also suppress STAT1 or IRF3 binding to the IFN-β promoter to inhibit type I IFN production (Huang et al., 2017).

3C of FMDV
Foot-and-mouth disease virus 3C plays important roles in disrupting the translational system of the host and can negatively regulate innate immune signaling by degrading essential molecules in different pathways (Ma et al., 2018a). FMDV 3C has the ability to cleave NEMO at Gln383 (Zhao et al., 2007); cleavage impairs NEMO-mediated IFN production and its ability to act as a signaling adaptor in the RIG-I/MDA5 pathway . Moreover, FMDV 3C cleaves TANK, generating a 15-kDa N-terminal fragment and impairing TANK's ability to suppress TRAF6-mediated NF-κB signaling .
Under normal conditions, ATG5-ATG12 promotes activation of IRF3 and phosphorylation of TBK1 by preventing TRAF3 degradation, resulting in enhanced expression of IFN-β . FMDV suppresses IRF3 by degradation of autophagy-related protein ATG5-ATG12 to attenuate production of IFN via 3C .
Karyopherin α1 (KPNA1) is the nuclear localization signal receptor for STAT1. FMDV 3C interferes with the JAK-STAT signaling pathway by degrading KPNA1, blocking STAT1/STAT2 nuclear translocation and inhibiting IFN signaling (Du et al., 2014). (Hales et al., 2008). SVV 3C has a conserved catalytic box with His and Cys residues (Qian et al., 2016), which is similar to FMDV Lpro. SVV 3C can inhibit the production of type I IFN by directly cleaving MAVS, TRIF, and TANK (Qian et al., 2016). In addition, a recent result indicates that SVV 3C reduces the expression of IRF3 and IRF7 and phosphorylates them and then blocks the transcription of IFN-β, IFN-α1, IFN-α4, and ISG54 (Xue et al., 2018).

3C of HAV
Hepatitis A virus 3C is a cysteine proteinase which is responsible for most cleavages within the viral polyprotein (Schultheiss et al., 1994(Schultheiss et al., , 1995. HAV 3C cleaves MAVS at Gln428 to inhibit type I IFN production . Similar to FMDV 3C, HAV 3C also cleaves NEMO, impairing NEMO-mediated IFN production and its ability to act as a signaling adaptor in the RIG-I/MDA5 pathway Xu et al., 2014). Moreover, HAV 3C inhibits NF-κB activation through cleavage of the TAK1/TAB1/TAB2/TAB3 complex, inhibiting the induction of IFNs (Lei et al., , 2014.

3ABC and 3CD of HAV
Processing intermediate HAV 3ABC and 3CD are both unique and have proteolytically activities in particle assembly (Probst et al., 1998). HAV 3ABC is a precursor cysteine protease. 3ABC cleaves MAVS and disrupts activation of IRF3 through the RLR pathway in mitochondria Debing et al., 2014). With the help of the transmembrane domain of 3A, 3ABC localizes to mitochondria. MAVS cleavage also requires the protease activity of 3C . This feature of 3ABC is unique among picornaviruses.

3D of EV71
Enterovirus 71 3D is a kind of RNA-dependent RNA polymerase (Jiang et al., 2011;Sun et al., 2012). Wang et al. (2015) found that without interfering with IFN-γ receptor expression, EV71 3D can attenuate STAT1 tyrosine phosphorylation resulting in defective IFN-γ signaling. The detailed signaling pathway how 3D regulate STAT1 need further investigation, Wang et al. (2015) guess that the function of EV71 3D may similar to EV71 2A, as a viral factor for immune-editing.

OUTLOOK
The interactions between picornaviruses and host defenses are complex and diverse. Moreover, viruses have developed multiple strategies to evade the host's innate immune system. To date, some of these strategies have been uncovered and significant progress has been achieved in understanding signaling pathways related to immune evasion. For example, the mechanism underlying inhibition by some non-structural proteins of IFN production in picornaviruses have been well studied. However, what we know today just represent a drop in the bucket, and we still need to understand the viral strategies involved in antagonizing the host's innate immune system. For example, SVV 3C has similar conserved catalytic box and similar function to FMDV Lpro in antagonizing the innate immune response and whether SVV 3C has other similar function to FMDV Lpro need further research. In addition, there are many similarities between different genera of picornaviruses. However, further efforts should be made to explore key mechanisms underlying inhibition by some non-structural proteins of IFN production, such as 2B, 2C, 3A, and 3D, across all picornavirus.
Recently, it has been discovered that some picornaviruses only cause an acute and self-limiting infection without major pathogenesis in hosts requiring more research on therapeutic approach (Weinberg and Morris, 2016;Ma et al., 2018b). The role of non-structural proteins in such picornaviruses may make contributions to better understand not only the therapeutic antiviral activity of IFNs, but also may reveal how these proteins (with or without protease activities) influence and control the IFN signaling transduction in vivo.

AUTHOR CONTRIBUTIONS
YW is the first author of this article and wrote the manuscript. LM, LS, and SS had made contributions to this article. XL and YL had revised this article.

FUNDING
This work was supported by Gansu Province Scientific and Technical Supporting Program (17YF1WA170).