key: cord-1033668-i8mh0uka
authors: Chin, Chue Vin; Saeed, Mohsan
title: Surgical Strikes on Host Defenses: Role of the Viral Protease Activity in Innate Immune Antagonism
date: 2022-04-28
journal: Pathogens
DOI: 10.3390/pathogens11050522
sha: 01015dc91c86558ee69a3d7a98c2fafa3fd0ff65
doc_id: 1033668
cord_uid: i8mh0uka

As a frontline defense mechanism against viral infections, the innate immune system is the primary target of viral antagonism. A number of virulence factors encoded by viruses play roles in circumventing host defenses and augmenting viral replication. Among these factors are viral proteases, which are primarily responsible for maturation of viral proteins, but in addition cause proteolytic cleavage of cellular proteins involved in innate immune signaling. The study of these viral protease-mediated host cleavages has illuminated the intricacies of innate immune networks and yielded valuable insights into viral pathogenesis. In this review, we will provide a brief summary of how proteases of positive-strand RNA viruses, mainly from the Picornaviridae, Flaviviridae and Coronaviridae families, proteolytically process innate immune components and blunt their functions.

Human cells are equipped to defend themselves against viral infections. When encountered by a virus, they mount a rapid and potent immune response that creates a protective environment in the infected cell and alerts the neighboring cells to an ongoing viral assault [1, 2] . A key to the success of this defense mechanism is an early detection of viral components, such as nucleic acids and proteins, collectively called pathogen-associated molecular patterns (PAMPs) [3] [4] [5] , and a quick relay of this information to immune effector molecules, which act as foot soldiers of the innate defense system and employ a variety of mechanisms to block viral propagation [1, 6, 7] . From detecting viral signatures to launching an effective counter-offensive against viral invasions, all steps in host cells are carried out by specialized proteins, some of which are constitutively expressed, while others are present at sub-detectable levels or exist in inactive forms during peacetime and only come into existence or action when the cell is under threat. A highly coordinated action of all of these proteins creates an intracellular environment averse to viral replication and spread.

Viruses, on the other hand, must circumvent host defenses to complete their lifecycle [8] . Therefore, it is no surprise that they have evolved a myriad of strategies to block innate immune signaling at almost every step along the way. These strategies include sequestration or degradation of host antiviral proteins [9] [10] [11] [12] [13] [14] , production of decoys to trick the immune system into chasing red herrings [15] [16] [17] , shielding viral components from immune surveillance [16, 18] , and suppressing the expression of antiviral genes [16, [19] [20] [21] [22] . A number of viruses, particularly those with a positive-sense RNA genome, encode proteases, necessitated by the requirement to cleave polyproteins generated during the lifecycle of these viruses. A large body of literature shows that viral proteases are key virulence factors that contribute to viral pathogenesis by limiting host responses [10, [23] [24] [25] . They do so by interacting with innate immune components in a non-catalytic fashion [9, [26] [27] [28] , proteolytically cleaving cellular proteins involved in innate immunity [10, 14, 25] , and altering protein Pathogens 2022, 11, 522 2 of 16 post-translational modifications critical for signal transduction [29] [30] [31] [32] [33] . This review focuses on proteolytic processing of host proteins and how it incapacitates the antiviral immune system. We begin by introducing proteases of three positive-strand RNA virus families, Picornaviridae, Flaviviridae, and Coronaviridae, followed by a brief description of key cellular proteins involved in innate immune signaling and their targeting by viral proteases.

Most positive-strand RNA viruses encode one or more proteases, which play key roles in maturation of viral proteins and inhibition of host antiviral functions. The role of the viral protease activity in host antagonism has been most extensively studied in the context of picornaviruses, flaviviruses, and coronaviruses. Picornaviruses are small non-enveloped viruses with a genome size between 7.5 and 10 kb and can be grouped into 68 genera (as of July 2021) [34] . The Enterovirus genus is the largest with several medically important members, such as poliovirus (PV), Coxsackieviruses (CVs), human rhinoviruses (HRVs), enterovirus D68 (EV-D68), EV-D70, and EV-A71. Other notable genera include Cardiovirus (containing encephalomyocarditis virus (EMCV)), Hepatovirus (containing hepatitis A virus (HAV)), and Aphthovirus (containing foot-and-mouth disease virus (FMDV)). The genome of these viruses encodes a single polyprotein of approximately 3000 amino acids length, which is processed by viral proteases into 11-12 mature proteins [35] [36] [37] ( Figure 1A) . The viral proteases involved in polyprotein processing include 2A pro and 3C pro for enteroviruses, L pro and 3C pro for aphthoviruses, and 3C pro for hepatoviruses and cardioviruses [38, 39] . The 2A pro and 3C pro are cysteine proteases that adopt the chymotrypsin-like fold [40] [41] [42] [43] , whereas L pro adopts the papain-like fold [44, 45] . P1 contains structural proteins, whereas P2 and P3 contain non-structural proteins. The first cleavage in the polyprotein is mediated by 2A pro , which cleaves at its N-terminus, separating P1 from P2-P3. P1 is further processed by 3C pro to yield three proteins, VP0, VP3, and VP1. Very late in infection, VP0 is cleaved into VP2 and VP4 by an unknown protease, although some studies suggest that it is an autocatalytic reaction [46, 47] . P2 and P3 are cleaved by 3C pro to liberate the total of seven non-structural proteins. (B) The polyprotein of the Flavivirus genus of the Flaviviridae family has two regions: One containing structural proteins and the other containing non-structural proteins. These proteins are liberated from the polyprotein through the action of host and viral proteases. Flaviviruses encode only one protease, NS2B-NS3 pro , that mediates six cleavages in the polyprotein. . P1 contains structural proteins, whereas P2 and P3 contain non-structural proteins. The first cleavage in the polyprotein is mediated by 2A pro , which cleaves at its N-terminus, separating P1 from P2-P3. P1 is further processed by 3C pro to yield three proteins, VP0, VP3, and VP1. Very late in infection, VP0 is cleaved into VP2 and VP4 by an unknown protease, although some studies suggest that it is an autocatalytic reaction [46, 47] . P2 and P3 are cleaved by 3C pro to liberate the total of seven non-structural proteins. The Flaviviridae family includes a number of clinically important viruses that mainly fall into two genera, Flavivirus and Hepacivirus. The various members of the genus Flavivirus include Zika virus (ZIKV), dengue virus (DENV), yellow fever virus (YF), West Nile virus (WNV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and tick-borne encephalitis virus (TBEV). These viruses are highly pathogenic to humans, mainly transmitted through mosquitoes and ticks, and prevalent in different parts of the world. The~11 kb genome of flaviviruses encodes a polyprotein that is cleaved by host proteases and a single viral protease, NS2B-NS3 pro , into 10 mature proteins [48, 49] ( Figure 1B) . The NS3 pro functions as an active enzyme only in the presence of the cofactor NS2B, which is an integral membrane protein of 14 kDa [48, 50, 51] : The NS3 protein is insoluble and catalytically inactive in the absence of NS2B, suggesting that NS2B has a role in NS3 folding [52] [53] [54] [55] . The genus Hepacivirus includes hepatitis C virus (HCV), which is responsible for an estimated 58 million cases of chronic hepatitis worldwide [56] . The 9.6 kb genome of this virus is translated into a single polyprotein, which is then processed by host and two viral proteases, NS2 pro and NS3-NS4A pro , to yield 10 mature proteins [57, 58] . NS3-NS4A pro , the main protease responsible for polyprotein processing, forms a noncovalent heterodimer consisting of a catalytic subunit NS3 and an activating cofactor NS4A [59, 60] . The NS2B-NS3 pro and NS3-NS4A pro are serine proteases with the chymotrypsin-like fold [61] [62] [63] .

The Coronaviridae family has recently gained global attention due to the unprecedented pandemic caused by the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [64, 65] . This virus was first identified in December 2019 [64] , and as of April 2022, has infected approximately 500 million individuals around the world and claimed around 6 million lives [66] . Before this, two other coronaviruses, SARS-CoV [67, 68] and Middle East respiratory syndrome coronavirus (MERS-CoV) [69] , made a zoonotic jump into humans and triggered large-scale outbreaks of severe respiratory disease during the past two decades. In addition to these highly pathogenic viruses, the Coronaviridae family also contains endemic viruses, such as NL-63, OC-43, 229-E, and HKU-1, which exhibit seasonality and mostly cause mild respiratory disease [70] . Coronaviruses have the largest genome (26-32 kb) of all known RNA viruses. The 5 -terminal two-third of the viral genome contains two open-reading frames (ORFs), 1a and 1b. ORF1a codes for polyprotein 1a, whereas ORF1a and 1b together encode polyprotein 1ab [71] . This latter mechanism is mediated by a (−1) ribosomal frameshift overreading the stop codon of ORF1a [72, 73] . The polyproteins 1a and 1ab are processed into 16 mature proteins by two viral proteases, papain-like protease (PL pro ) and a 3C-like protease (3CL pro ) [74, 75] ( Figure 1C ). Both PL pro and 3CL pro are cysteine proteases, however, while PL pro adopts the papain-like fold [76, 77] , 3CL pro features the chymotrypsin-like fold [78, 79] .

Beside their role in maturation of viral polyproteins, viral proteases cleave host proteins, particularly those involved in innate antiviral immunity (Figure 2 ), thereby neutralizing host defenses and creating an environment that favors virus replication. In this section, we will first provide a brief overview of cellular proteins that perform various functions in innate immune defenses and then present examples from the literature illustrating viral protease-mediated cleavage of these proteins.

Innate immune signaling begins with the detection of PAMPs, either on the cell surface or within cellular compartments, through specialized host proteins, called pattern recognition receptors (PRRs) [80] . The PRRs involved in the recognition of positive-strand RNA viruses can be divided into two broad categories according to the cellular area they surveil. The first category consists of toll-like receptors (TLRs), which reside on the cell surface and in endosomes and sense extracellular and endosome-localized viral signatures [81, 82] . The TLR family comprises 10 members (TLR1-10) in humans [83] , of which TLR3, TLR7, and TLR8 have been implicated in detection of viral RNA. TLR3 recognizes double-stranded RNA, whereas TLR7 and 8 detect single-stranded RNA [84] . The second group of PRRs include RIG-I-like receptors (RLRs) [85] and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) [86] , which recognize viral nucleic acids in the cytoplasm of infected cells. Three major RLRs identified to date are RIG-I [87] , MDA5 [88] , and LGP2 [89] , which all sense viral RNA, but exhibit differences in their recognition specificity and functional characteristics. Twenty-three NLR proteins have been described in humans [90, 91] . Among these, NOD2 has been shown to recognize viral RNA and induce antiviral responses in infected cells [92] .

ization domain (NOD)-like receptors (NLRs) [86] , which recognize viral nucleic acids in the cytoplasm of infected cells. Three major RLRs identified to date are RIG-I [87] , MDA5 [88] , and LGP2 [89] , which all sense viral RNA, but exhibit differences in their recognition specificity and functional characteristics. Twenty-three NLR proteins have been described in humans [90, 91] . Among these, NOD2 has been shown to recognize viral RNA and induce antiviral responses in infected cells [92] . [93] . These sensors then interact with downstream adaptor proteins to initiate multiple signaling cascades. TLR7/8 and TLR9 employ MyD88 as an adaptor, whereas TLR3 signals through the TRIF adaptor. RIG-I and MDA5 use MAVS as an adaptor protein, although RIG-I has been reported to also signal through STING. The adaptor proteins, once bound by immune sensors, assemble large signaling complexes containing several cellular proteins, ultimately activating two major transcription factors, NF-κB and IRFs. These transcription factors travel to the nucleus and induce the expression of genes encoding proinflammatory cytokines, IFNs, and ISGs. IFNs are released into the extracellular space, where they bind to their respective cell surface receptors and trigger phosphorylation-dependent activation of pre-associated receptor tyrosine kinases, such as JAK1 and TYK2 [94] . This leads to recruitment and phosphorylation of STAT proteins. STAT1 and STAT2 form a heterodimer, which in turn recruits IRF9 to form the ISGF3 complex. This complex translocates to the nucleus and binds ISRE promoter elements, inducing the expression of ISGs. Viruses that employ their proteases to target various innate immune components are shown in red.

Viruses target PRRs to evade innate immune surveillance. Due to their localization on the cell surface and in endosomal compartments, TLRs mostly remain shielded from exposure to viral proteases. The RLRs, on the other hand, reside in the cytoplasm and are frequently targeted by viral proteases. The Coxsackievirus B3 (CVB3) 2A pro and FMDV L pro have been shown to cleave MDA5 [95, 96] . The cleavage occurs at a highly conserved Figure 2 . Antiviral innate immune pathways and their targeting by viral proteases. PAMPS associated with positive-strand RNA viruses are sensed by TLR3, TLR7/8, and TLR9 in endosomes and by RIG-I and MDA5 in the cytoplasm [93] . These sensors then interact with downstream adaptor proteins to initiate multiple signaling cascades. TLR7/8 and TLR9 employ MyD88 as an adaptor, whereas TLR3 signals through the TRIF adaptor. RIG-I and MDA5 use MAVS as an adaptor protein, although RIG-I has been reported to also signal through STING. The adaptor proteins, once bound by immune sensors, assemble large signaling complexes containing several cellular proteins, ultimately activating two major transcription factors, NF-κB and IRFs. These transcription factors travel to the nucleus and induce the expression of genes encoding proinflammatory cytokines, IFNs, and ISGs. IFNs are released into the extracellular space, where they bind to their respective cell surface receptors and trigger phosphorylation-dependent activation of pre-associated receptor tyrosine kinases, such as JAK1 and TYK2 [94] . This leads to recruitment and phosphorylation of STAT proteins. STAT1 and STAT2 form a heterodimer, which in turn recruits IRF9 to form the ISGF3 complex. This complex translocates to the nucleus and binds ISRE promoter elements, inducing the expression of ISGs. Viruses that employ their proteases to target various innate immune components are shown in red.

Viruses target PRRs to evade innate immune surveillance. Due to their localization on the cell surface and in endosomal compartments, TLRs mostly remain shielded from exposure to viral proteases. The RLRs, on the other hand, reside in the cytoplasm and are frequently targeted by viral proteases. The Coxsackievirus B3 (CVB3) 2A pro and FMDV L pro have been shown to cleave MDA5 [95, 96] . The cleavage occurs at a highly conserved RGRAR motif and renders MDA5 non-functional by separating the viral RNA-binding C-terminal domain (CTD) from the two signal-transducing N-terminal caspase activation and recruitment domains (CARDs) that mediate interaction with the downstream adaptor proteins [96] . Notably, MDA5 is also bound by 3C pro of several picornaviruses, such as Coxsackievirus-A6 (CV-A6), CV-A16, EV-D68, and EV-A71. However, this interaction does Pathogens 2022, 11, 522 6 of 16 not involve MDA5 cleavage, but instead limits the downstream interaction of MDA5 with MAVS [28, 97] .

RIG-I is also targeted by viral proteases. The 3C pro of EV-A71, CVB3, PV, and EMCV has been reported to inhibit RIG-I [95, 98] . Conflicting data exist about the mechanisms that underlie 3C pro -mediated RIG-I inhibition. While There is not much literature on LGP2 processing by viral proteases. The FMDV L pro has been reported to cleave LGP2 [96] . As with MDA5, the protease targets the RGRAR sequence in the conserved helicase motif of LGP2, yielding protein products that can no longer regulate antiviral immunity in infected cells.

Upon binding with PAMPs, the PRRs get activated and engage specific adaptor proteins, which serve as scaffolds for the assembly of large signaling complexes, called signalosomes. These signaling bodies then transduce signals to the downstream effector proteins. The TLRs signal via two major adapters, MyD88 and TRIF, leading to activation of transcription factors NF-κB and interferon-regulatory factors (IRFs) [101] [102] [103] [104] [105] . The MyD88-dependent pathway operates through phosphorylation-dependent degradation of the NF-κB inhibitory protein IκBα [104, 106] . In unstimulated cells, NF-κB resides in the cell cytoplasm in an inhibitory complex with IκBα [107] . Activation of the MyD88 pathway and subsequent degradation of IκBα liberates NF-κB, which then migrates to the nucleus and induces the expression of proinflammatory genes. The TRIF-dependent pathway stimulates IRF3 and NF-κB signaling cascades, leading to expression of proinflammatory genes and type I interferon (IFN) [101, 108, 109] .

The RLRs signal through the adaptor protein, MAVS (also known as IPS-1/VISA/ Cardif) [110] [111] [112] [113] , that rapidly recruits a large number of signaling proteins to activate IRF and NF-κB pathways [111] . MAVS has three functional domains; an N-terminal CARD domain for interaction with immune sensors, a C-terminal transmembrane domain for localization to distinct cellular places, such as mitochondrial membranes, peroxisomes, and mitochondrial-associated membranes, and a proline-rich domain for engagement with functional partners [111, [114] [115] [116] [117] [118] . Based on the composition of the MAVS signalosome, the RLR signaling bifurcates into two molecular cascades. The first cascade involves a protein complex of Tank binding kinase-1 (TBK1) and IκB kinase epsilon (IKKε), which directly phosphorylates IRF3 and IRF7 to induce the expression of IFN genes. The second cascade recruits the IKKα/β/γ complex (IKKγ is also known as NEMO) to cause phosphorylation and proteasomal degradation of IκBα, liberating NF-κB from the NF-κB-IκBα complex. The NF-κB then translocates to the nucleus and induces the expression of proinflammatory cytokines [119, 120] .

Another protein, stimulator of IFN gene (STING; also known as MITA, MPYS, and ERIS) [121] [122] [123] [124] , which mainly serves as an adaptor in the DNA sensing pathway, has been shown to participate in the transmission of RIG-I, but not MDA5, signals [122, 125] . This role of STING is mediated by its direct interaction with RIG-I and MAVS in a complex that is stabilized upon virus infection, leading to activation of IRFs [122, 123, 125] . STING is also activated by the mitochondrial DNA being released into the cytoplasm of RNA virus-Pathogens 2022, 11, 522 7 of 16 infected cells, which is then detected by the canonical DNA sensor, cGAS [126] , triggering downstream activation of STING and subsequent production of IFN [127] [128] [129] .

The adaptor proteins represent central signaling hubs of antiviral networks and are therefore an attractive target of virus-mediated proteolysis. MAVS is probably the most well characterized target of viral proteases. It is cleaved by a number of proteases from diverse virus families. Picornaviral proteases, including those of CVB3, PV, rhinoviruses, EV-D70, and EV-A71 [14, 95, 130] , are known to cleave MAVS, with the cleavage site varying between viruses [14] . While these cleavages are mainly carried out by 2A pro [95, 131, 132] , L pro and 3C pro of some picornaviruses, such as CVB, Seneca Valley virus (SVV), and FMDV, have also demonstrated the ability to cleave MAVS [130, 133, 134] . Another picornavirus, HAV, employs a 3C pro precursor, 3ABC pro , to cleave MAVS [100] . The transmembrane domain of 3A enables 3ABC to anchor on the mitochondrial membrane, and the protease activity of 3C pro then carries out the MAVS cleavage. The mature 3C pro alone does not localize to mitochondria and is therefore incapable of targeting MAVS. The HCV NS3-NS4A pro also cleaves MAVS, leading to suppression of the innate immune pathway [112, 135, 136] . Mechanistically, in all cases of viral protease-mediated cleavage, the CARD domain of MAVS is dislodged from the mitochondrial membrane, thereby blocking formation of the MAVS signalosome and disrupting signal transduction.

TRIF is also a common target of viral proteases. Both 2A pro and 3C pro of picornaviruses have been reported to process TRIF [130, 132, 137, 138] . However, like MAVS, TRIF also seems to be cleaved at different sites by different viruses; while the EV-71 3C pro cleaves TRIF at only one site [138] , the EV-D68 3C pro cleaves at two sites [137] . It is currently unknown if these differential cleavage patterns are linked to differences in the biological response. Interestingly, HAV protease-polymerase precursor 3CD pro , but not the mature 3C pro , cleaves TRIF [99] . The TRIF cleavage has also been reported for HCV NS3-NS4A pro [139] .

Several positive-strand RNA viruses employ their proteases to target the STING protein, authenticating the relevance of this DNA sensing adapter to RNA viruses. Many flaviviruses, such as ZIKV, DENV, WNV, and JEV, but not YFV, use their NS2B-NS3 pro to cleave the human STING protein [140] [141] [142] [143] . Notably, these viruses do not cleave mouse STING, and this deficiency has been linked, at least for ZIKV, to restricted viral replication in murine cells; knocking out the expression of STING in mouse cell lines caused~10-50-fold increase in ZIKV replication [140] . Similarly, the DENV NS2B-NS3 pro , although efficiently cleaves human STING, does not target the STING versions found in most other mammals, including non-human primates, possibly explaining the differential replication levels achieved by DENV in human versus other species [143] .

The signaling cascades unleashed upon sensing of viral PAMPs in infected cells lead to production of proinflammatory cytokines and IFNs. Two major transcription factors that regulate induction of these genes are NF-κB and IRFs [144, 145] . The NF-κB family comprises five structurally related members, including NF-κB1 (also known as p50), NF-κB2 (also known as p52), RelA (also known as p65), RelB, and c-Rel [146] . These proteins, paired in distinct homo-and heterodimers, bind to a specific DNA element, κB enhancer, to mediate the transcription of target genes [147] . As described above, the NF-κB proteins normally exist in a latent form in unstimulated cells through binding to members of the IκB family of inhibitory proteins, mainly IκBα, which masks the nuclear localization signal of associated NF-κB proteins. Activation of immune signaling causes degradation of IκBα, triggered through its site-specific phosphorylation by a multisubunit IκB kinase (IKK) complex [146, 148] . IKK has two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, NEMO (also called IKKγ) [149] . Once activated, IKK phosphorylates IκBα at two N-terminal serines and triggers ubiquitination-dependent IκBα degradation [107, 148] , allowing rapid and transient translocation of NF-κB members, predominantly the p50/RelA and p50/c-Rel dimers, to the nucleus, where they bind κB enhancers and induce the transcription of hundreds of target genes, most of which encode proinflammatory cytokines [107, 146, 148, [150] [151] [152] [153] .

The second group of transcription factors are IRFs that are primarily involved in transcription of IFN and IFN-stimulated genes (ISGs). Since the discovery of the first IRF, IRF1, in 1988 [154, 155] , the total of nine IRFs (IRF1-9) have been characterized in mammals. They bind to a specific DNA motif, called IFN-stimulated response element (ISRE), as distinct homo-and hetero-dimers and regulate the expression of a large cohort of genes [156] . Apart from interacting among themselves, IRFs also partner with other transcription factors, such as STATs and NF-κB, activating a broad spectrum of genes and controlling diverse transcriptional programs [157] . While all IRFs play roles in immune response of cells, IRF3 and IRF7 are the crucial modulators of IFN production [158] [159] [160] . Both IRF3 and IRF7 exist in an inactive form in the cytoplasm of uninfected cells [161, 162] . Upon virus infection, they are activated through TBK1 and IKKε-mediated phosphorylation [163] [164] [165] [166] , which induces conformational changes in IRFs, facilitating their dimerization and subsequent nuclear translocation, where, along with other co-activators, they regulate transcription of type I IFN and ISGs [1, 167] .

IRFs and NF-κB are both targeted by viral proteases. EV-A71 and EV-D68 employ their 3C pro to process IRF7 [168, 169] , the main transcription factor involved in IFNα production. While EV-A71 3C pro cleaves IRF7 only at one site [168] , EV-D68 3C pro processes the protein at two sites [169] ; however, the functional consequences of these differential cleavages are unknown. Another picornavirus, SVV, has been reported to reduce cellular levels of both IRF3 and IRF7 in a manner that depends on the enzymatic activity of the viral 3C pro [170] . A recently published study demonstrated the ability of the SARS-CoV-2 PL pro to cleave IRF3 [171] . The NF-κB family members are also proteolytically processed by viral proteases. The 3C pro of PV, ECHO-1, and human rhinovirus B-14 cleaves the p65 subunit of the NF-κB complex, thereby shutting down the induction of proinflammatory genes [172] . In addition to direct cleavage, viral proteases can also block the nuclear migration of transcription factors by targeting the protein transport machinery. In this vein, HCV NS3-NS4A pro has been shown to inhibit IFN production by cleaving importin β1, a key nucleocytoplasmic transport receptor involved in nuclear import of IRF3 and NF-κB [173] .

The IFN family includes three main classes of related cytokines: Type I IFNs, type II IFN, and type III IFNs. Once released from virus-infected cells, they work in an autocrine and paracrine manner to create an antiviral state in both infected cells and the neighboring bystander cells. Interaction of IFNs with their receptors triggers activation of receptor tyrosine kinases JAK1 and TYK2, which in turn phosphorylate members of the STAT family of proteins, triggering their dimerization [174] . STAT1 and STAT2 are the main IFN-activated transcription factors, which, together with IRF9, form a trimeric complex, ISGF3, that travels to the nucleus and drives transcription of ISGs [175] . These ISGs, which are several hundred in number, are the workhorse of the innate immune system and employ a variety of mechanisms to block and/or eliminate viral infections [6, 7] .

In addition to ablating the IFN production pathway, viral proteases also block events that happen after IFNs bind to their receptors on the cell surface. In an elegant study, Morrison et al. showed that several picornaviruses, such as EV-D70, HRV A16, and PV, are capable of replicating in type I IFN-treated cells in a 2A pro -dependent manner [176] .

While mechanisms through which 2A pro limits the antiviral effect of IFN are unknown, the authors speculated that the protease might be cleaving ISGs responsible for inhibition of picornavirus replication. In another study, the EV-A71 3C pro was shown to target IRF9 for proteolytic cleavage [177] , abrogating the ability of IFN to inhibit viral replication. Porcine deltacoronavirus (PDCoV) has been reported to employ its 3CL pro to cleave STAT2 and inhibit the ISRE reporter activity [178] .

This review summarized the role of the viral protease activity in counteracting host defenses. Historically, the study of virus-mediated proteolysis of host proteins has relied on targeted molecular biology techniques, such as western blot. However, with terrific advances in systems biology approaches, it has now become possible to perform a global survey of protein cleavages in virus-infected cells [14, 179] . Defining the full extent of proteolysis in virus-infected cells and performing the functional characterization of newly identified host substrates can illuminate the complexities of the innate immune system and highlight the role of viral proteases as important virulence factors. Further, most published studies have only utilized mature forms of viral proteases to test the cleavage of a cellular protein, running the risk of yielding false-negative results. There are examples when the precursor forms of a viral protease, but not its mature form, cleaves a host protein [99, 100] . This could be due to altered subcellular localization of the precursor form or changes in the contours of the protease active site, modulating the affinity of substrate binding and thereby influencing the substrate cleavage efficiency or specificity. Therefore, unless a protein is tested in virus-infected cells, the possibility of it being cleaved by a viral protease should not be ruled out. Moreover, in some cases, only a small fraction of a given cellular protein is being targeted for cleavage. For example, while the cleavage of MAVS in picornavirusinfected cells disrupts antiviral signaling, the total amount of full-length MAVS in virusinfected cells appears to remain relatively unchanged when tested by western blot [14] . This can be attributed to a possibility that, at least in some cases, viral proteases target only the portion of a host protein localized to a particular subcellular organelle. For instance, it is conceivable that only the fraction of MAVS localized to mitochondrial membranes or peroxisomes is being targeted by a viral protease. With rapidly improving performance of proteomic approaches and increasing interest in viral proteases as attractive drug targets, it should soon become possible to define precise spatiotemporal regulation of protein cleavages in virus-infected cells. 

The authors declare no conflict of interest.

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The structure of the 2A proteinase from a common cold virus: A proteinase responsible for the shut-off of host-cell protein synthesis

Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein

Structure-Based Design of Michael Acceptor Inhibitors and Their Broad-Spectrum Antiviral Effects against Picornaviruses

The structures of picornaviral proteinases

Structure of the foot-and-mouth disease virus leader protease: A papain-like fold adapted for self-processing and eIF4G recognition

Three-Dimensional Structure of Poliovirus at 2.9 Å Resolution

Structure of a human common cold virus and functional relationship to other picornaviruses

Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins

Production of yellow fever virus proteins in infected cells: Identification of discrete polyprotein species and analysis of cleavage kinetics using region-specific polyclonal antisera

Cotranslational Membrane Insertion of the Serine Proteinase Precursor NS2B-NS3(Pro) of Dengue Virus Type 2 Is Required for Efficient in Vitro Processing and Is Mediated through the Hydrophobic Regions of NS2B

Processing of Japanese encephalitis virus non-structural proteins: NS2B-NS3 complex and heterologous proteases

Structure of the Dengue Virus Helicase/Nucleoside Triphosphatase Catalytic Domain at a Resolution of 2.4 Å

NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex

Dengue protease activity: The structural integrity and interaction of NS2B with NS3 protease and its potential as a drug target

Activation of dengue protease autocleavage at the NS2B-NS3 junction by recombinant NS3 and GST-NS2B fusion proteins

Global Progress Report on HIV, Viral Hepatitis and Sexually Transmitted Infections

Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase

Overview of Hepatitis C Virus Genome Structure, Polyprotein Processing, and Protein Properties

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism

Mechanistic role of NS4A and substrate in the activation of HCV NS3 protease

Crystal Structure of the Hepatitis C Virus NS3 Protease Domain Complexed with a Synthetic NS4A Cofactor Peptide

Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus

Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Wuhan to World: The COVID-19 Pandemic

Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome

A Novel Coronavirus Associated with Severe Acute Respiratory Syndrome

Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia

Global seasonality of human coronaviruses: A systematic review. Open Forum Infect

Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach

A Three-Stemmed mRNA Pseudoknot in the SARS Coronavirus Frameshift Signal

Characterization of an efficient coronavirus ribosomal frameshifting signal: Requirement for an RNA pseudoknot

Processing of the Human Coronavirus 229E Replicase Polyproteins by the Virus-Encoded 3C-Like Proteinase: Identification of Proteolytic Products and Cleavage Sites Common to pp1a and pp1ab

The SARS-coronavirus papain-like protease: Structure, function and inhibition by designed antiviral compounds

Structural and mutational analysis of the interaction between the Middle-East respiratory syndrome coronavirus (MERS-CoV) papain-like protease and human ubiquitin

Crystal structure of the papain-like protease of MERS coronavirus reveals unusual, potentially druggable active-site features

Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain

From SARS to MERS: Crystallographic studies on coronaviral proteases enable antiviral drug design

Pattern recognition receptors in health and diseases

Toll-like receptors and innate immunity

Toll-like receptors and innate immunity

Quantitative Expression of Toll-Like Receptor 1-10 mRNA in Cellular Subsets of Human Peripheral Blood Mononuclear Cells and Sensitivity to CpG Oligodeoxynucleotides

The role of pattern-recognition receptors in innate immunity: Update on Toll-like receptors

RIG-I-like receptors: Their regulation and roles in RNA sensing

Functions of NOD-Like Receptors in Human Diseases

The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses

mda-5: An interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties

The RNA Helicase Lgp2 Inhibits TLR-Independent Sensing of Viral Replication by Retinoic Acid-Inducible Gene-I

NOD-like receptors (NLRs): Bona fide intracellular microbial sensors

The NOD: A signaling module that regulates apoptosis and host defense against pathogens

Activation of innate immune antiviral responses by Nod2

Pattern Recognition Receptors and the Innate Immune Response to Viral Infection

Mechanisms of type-I-and type-II-interferon-mediated signalling

Enterovirus 2A pro Targets MDA5 and MAVS in Infected Cells

MDA5 cleavage by the Leader protease of foot-and-mouth disease virus reveals its pleiotropic effect against the host antiviral response

The 3C Protein of Enterovirus 71 Inhibits Retinoid Acid-Inducible Gene I-Mediated Interferon Regulatory Factor 3 Activation and Type I Interferon Responses

The viral RNA recognition sensor RIG-I is degraded during encephalomyocarditis virus (EMCV) infection

Disruption of TLR3 Signaling Due to Cleavage of TRIF by the Hepatitis A Virus Protease-Polymerase Processing Intermediate, 3CD

Disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor

Role of Adaptor TRIF in the MyD88-Independent Toll-Like Receptor Signaling Pathway

MyD88: A central player in innate immune signaling

MyD88: A Critical Adaptor Protein in Innate Immunity Signal Transduction

Signaling to NF-κB by Toll-like receptors

Toll-like Receptor and RIG-1-like Receptor Signaling

Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway

Regulation and Function of IKK and IKK-Related Kinases

Cutting Edge: A Novel Toll/IL-1 Receptor Domain-Containing Adapter That Preferentially Activates the IFN-β Promoter in the Toll-Like Receptor Signaling

Lipopolysaccharide Stimulates the MyD88-Independent Pathway and Results in Activation of IFN-Regulatory Factor 3 and the Expression of a Subset of Lipopolysaccharide-Inducible Genes

IPS-1, an adaptor triggering RIG-I-and Mda5-mediated type I interferon induction

Identification and Characterization of MAVS, a Mitochondrial Antiviral Signaling Protein that Activates NF-κB and IRF3

Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus

VISA Is an Adapter Protein Required for Virus-Triggered IFN-β Signaling

MAVS Coordination of Antiviral Innate Immunity

Prion-like Polymerization Underlies Signal Transduction in Antiviral Immune Defense and Inflammasome Activation

MAVS Self-Association Mediates Antiviral Innate Immune Signaling

Peroxisomes Are Signaling Platforms for Antiviral Innate Immunity

Mitochondrial-associated endoplasmic reticulum membranes (MAM) form innate immune synapses and are targeted by hepatitis C virus

RNA recognition and signal transduction by RIG-I-like receptors

RIG-I-like receptors: Sensing and responding to RNA virus infection

ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization

The Adaptor Protein MITA Links Virus-Sensing Receptors to IRF3 Transcription Factor Activation

STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling

MPYS, a Novel Membrane Tetraspanner, Is Associated with Major Histocompatibility Complex Class II and Mediates Transduction of Apoptotic Signals

Mediates Neuronal Innate Immune Response Following Japanese Encephalitis Virus Infection

Cyclic GMP-AMP Synthase Is a Cytosolic DNA Sensor That Activates the Type I Interferon Pathway

Dengue virus activates cGAS through the release of mitochondrial DNA

The interactions between cGAS-STING pathway and pathogens

Collateral Damage during Dengue Virus Infection: Making Sense of DNA by cGAS

The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling

Enterovirus 71 Protease 2Apro Targets MAVS to Inhibit Anti-Viral Type I Interferon Responses

Coxsackievirus counters the host innate immune response by blocking type III interferon expression

Seneca Valley Virus Suppresses Host Type I Interferon Production by Targeting Adaptor Proteins MAVS, TRIF, and TANK for Cleavage

Dissecting distinct proteolytic activities of FMDV Lpro implicates cleavage and degradation of RLR signaling proteins, not its deISGylase/DUB activity, in type I interferon suppression

Cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis C correlates with a reduced activation of the endogenous interferon system

Dissociation of a MAVS/IPS-1/VISA/Cardif-IKKε Molecular Complex from the Mitochondrial Outer Membrane by Hepatitis C Virus NS3-4A Proteolytic Cleavage

Enterovirus 68 3C Protease Cleaves TRIF To Attenuate Antiviral Responses Mediated by Toll-Like Receptor 3

Cleavage of the Adaptor Protein TRIF by Enterovirus 71 3C Inhibits Antiviral Responses Mediated by Toll-Like Receptor 3

Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF

PLoss, A. Species-specific disruption of STING-dependent antiviral cellular defenses by the Zika virus NS2B3 protease

DENV Inhibits Type I IFN Production in Infected Cells by Cleaving Human STING

Dengue Virus Targets the Adaptor Protein MITA to Subvert Host Innate Immunity

Dengue viruses cleave STING in humans but not in nonhuman primates

The Role of Nuclear Factor κB in the Interferon Response

The IRF family of transcription factors: Inception, impact and implications in oncogenesis

The NF-B Family of Transcription Factors and Its Regulation

Regulation of nuclear factor-κB in autoimmunity

The IκB kinase (IKK) and NF-κB: Key elements of proinflammatory signalling

New insights into NF-κB regulation and function

Functions of NF-κB1 and NF-κB2 in immune cell biology

Shared Principles in NF-κB Signaling

Activators and target genes of Rel/NF-kappaB transcription factors

Crystal structure of p50/p65 heterodimer of transcription factor NF-κB bound to DNA

Evidence for a nuclear factor(s), IRF-1, mediating induction and silencing properties to human IFN-beta gene regulatory elements

Regulated expression of a gene encoding a nuclear factor, IRF-1, that specifically binds to IFN-β gene regulatory elements

Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins

Regulatory Networks Involving STATs, IRFs, and NFκB in Inflammation

Distinct and Essential Roles of Transcription Factors IRF-3 and IRF-7 in Response to Viruses for IFN-α/β Gene Induction

IRF-7 is the master regulator of type-I interferon-dependent immune responses

An Atomic Model of the Interferon-β Enhanceosome

Structural and Functional Analysis of Interferon Regulatory Factor 3: Localization of the Transactivation and Autoinhibitory Domains

Multiple Regulatory Domains Control IRF-7 Activity in Response to Virus Infection

Activation of TBK1 and IKKε Kinases by Vesicular Stomatitis Virus Infection and the Role of Viral Ribonucleoprotein in the Development of Interferon Antiviral Immunity

Contribution of a TANK-Binding Kinase 1-Interferon (IFN) Regulatory Factor 7 Pathway to IFN-γ-Induced Gene Expression

IKKε and TBK1 are essential components of the IRF3 signaling pathway

Triggering the Interferon Antiviral Response Through an IKK-Related Pathway

IRF-3, IRF-5, and IRF-7 Coordinately Regulate the Type I IFN Response in Myeloid Dendritic Cells Downstream of MAVS Signaling

Cleavage of Interferon Regulatory Factor 7 by Enterovirus 71 3C Suppresses Cellular Responses

3C Protease of Enterovirus D68 Inhibits Cellular Defense Mediated by Interferon Regulatory Factor 7

Seneca Valley Virus 3Cpro abrogates the IRF3-and IRF7-mediated innate immune response by degrading IRF3 and IRF7

SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): Implications for disease presentation across species

Proteolytic Cleavage of the p65-RelA Subunit of NF-κB during Poliovirus Infection

Importin β1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF-κB signaling of IFNB1 antiviral response

How cells respond to interferons

From Interferons to Cytokines

Proteinase 2A pro Is Essential for Enterovirus Replication in Type I Interferon-Treated Cells

Synergistic Inhibition of Enterovirus 71 Replication by Interferon and Rupintrivir

Porcine Deltacoronavirus nsp5 Antagonizes Type I Interferon Signaling by Cleaving STAT2

Characterising proteolysis during SARS-CoV-2 infection identifies viral cleavage sites and cellular targets with therapeutic potential