Trends in

Immunology
Review

Effector-triggered immunity in mammalian

antiviral defense
1,
Megan H. Orzalli * and Pooja Parameswaran1

Effector-triggered immunity (ETI) is a common defense strategy used by mam- Highlights

malian host cells that is engaged upon detection of the enzymatic activities of SARS-CoV-2 viroporins and NSP5 pro-
pathogen-encoded proteins or the effects of their expression on cellular homeo- tease activities are sensed by human
cells to trigger inflammasome-mediated
stasis. However, in contrast to the effector-triggered responses engaged upon
pyroptosis.
bacterial infection, much less is understood about the activation and conse-
quences of these responses following viral infection. Several recent studies Guard proteins monitor viral inhibition of
have identified novel mechanisms by which viruses engage ETI, highlighting host cell protein synthesis within barrier
epithelia and contribute to antiviral de-
the importance of these immune responses in antiviral defense. We summarize fense against diverse viruses in human
recent advances in understanding how mammalian cells sense virus-encoded skin.
effector proteins, the downstream signaling pathways that are triggered by
these sensing events, and how viruses manipulate these pathways to become A novel self-guarded signaling path-
way induces IFNB expression when
more successful pathogens. disrupted and contributes to antiviral
defense against herpes simplex virus
1 in human monocytes.

An overview of antimicrobial innate immunity

Mammalian antimicrobial defense relies on a combination of physical barriers, rapid activation of Significance
innate immunity, and the production of adaptive immune responses. The best-studied antiviral The role of effector-triggered immunity
innate immune mechanisms used by mammalian cells are those engaged by germline-encoded in mammalian host defense against
virus infection is poorly characterized.
pattern recognition receptors (PRRs; see Glossary) that detect pathogen-associated However, recent studies have identi-
molecular patterns (PAMPs) contained within the virion or that are produced following infection. fied new ways by which host cells
Viral genomes or replication intermediates are a major source of PAMPs and are sensed in recognize viral effectors, highlighting
mammalian cells by PRRs including RIG-I, MDA5, TLR3, TLR9, TLR7, IFI16 and cGAS [1]. Upon the importance of this host defense
strategy in antiviral immunity.
PAMP recognition, PRRs engage downstream signaling pathways resulting in the activation and
nuclear translocation of transcription factors including IRF3, NF-κB, and AP-1 [1]. Consequently,
these transcription factors induce type I and type III interferon (IFN) cytokines that promote the
expression of antiviral IFN-stimulated genes [2]. Further, nucleic acid-sensing PRRs such as
AIM2 and ZBP1 can activate the proinflammatory cell death pathways pyroptosis and
necroptosis, respectively [3]. Collectively, these antimicrobial responses that rely on the detection
of PAMPs are known as pattern-triggered immunity (PTI).

Pathogens produce effector proteins that disrupt PTI, potentially leaving the host susceptible
to infection [4]. However, hosts also use effector-triggered immunity (ETI) to activate 1
Program in Innate Immunity, Division of
antipathogen defenses when PTI is inhibited. In contrast to PTI, ETI surveils for pathogen effector Infectious Diseases and Immunology,
activities rather than PAMPs. Indirect mechanisms of ETI, such as those provided by guard and Department of Medicine, University of
Massachusetts Chan Medical School,
decoy proteins, are prevalent in mammals and are vital for sensing pathogenic bacteria [5]. In the
Worcester, MA, USA
'guard' model, host proteins (guards) monitor cellular pathways (guardees) and initiate immune
responses when these pathways are perturbed [6]. In mammals, this model is exemplified by
the Pyrin guard protein which senses inactivation of the guardee RhoA by the Clostridium difficile
TcdB effector [7]. Further, decoy proteins, such murine Nlrp1b that is triggered by anthrax lethal
*Correspondence:
toxin and Shigella flexinari IpaH7.8 [8,9], are cellular proteins that commonly recognize the megan.orzalli@umassmed.edu
enzymatic activities (e.g., protease activity, ubiquitin ligase activity) of pathogen effectors. Importantly, (M.H. Orzalli).

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© 2022 Elsevier Ltd. All rights reserved.
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these decoy proteins themselves do not contain antipathogen activities, but instead mimic host de- Glossary
fense components and engage innate immune responses when accidentally targeted by effectors [5]. Apoptosis: an immunologically silent
caspase-dependent cell-death
pathway. Can be activated through
In contrast to the role ETI plays in antibacterial defense, its role in antiviral defense remains less
extrinsic (e.g., death receptors) or
well studied. However, it is becoming apparent that mammalian cells sense viral effectors and intrinsic (e.g., mitochondrial) stimuli.
that ETI contributes to antiviral defense. In this review we highlight recent studies describing the ASC: an adaptor protein that forms a
mechanisms by which viruses engage ETI, including the sensing of virus-induced changes in multimeric complex with inflammasome
proteins and executor caspases during
cellular ion gradients, viral protease activity, virus-mediated inhibition of host cell protein synthesis
pyroptosis (apoptosis-associated
that triggers pyroptosis, and perturbations in self-guarded protein pathways. Understanding speck-like protein containing a CARD)
the contribution of ETI to antiviral defense is important because it may provide insight into defense that can be visualized by microscopy as
strategies that might be targeted for therapeutic interventions during infection. a speck.
Damage-associated molecular
patterns (DAMPs): molecules
Sensing virus-induced disruptions in cellular ion gradients released upon cell stress, injury, or
Pyroptosis is a common consequence of activating ETI. This can occur through the formation of death, leading to innate immune
inflammasomes, which are large cytosolic protein complexes that activate inflammatory responses in bystander cells.
Effector-triggered immunity (ETI):
proteases such as caspase-1 [10]. The best-studied inflammasome is that seeded by the immune responses triggered by
nucleotide-binding domain, leucine-rich repeat (NLR) family, pyrin domain (PYD)-containing protein pathogen-encoded proteins that
3 (NLRP3), which complexes with the adaptor protein ASC to recruit and activate caspase-1 in change cellular homeostasis.
mammalian cells [11]. Active caspase-1 cleaves the proinflammatory cytokines IL-1β and IL-18, as Humanized mouse model of SARS-
CoV-2 infection: humanized MISTRG6
well as the pore-forming protein gasdermin D (GSDMD) which provides a conduit across the plasma mice which ACE2 is delivered by
membrane for IL-1 cytokine release [12]. In addition, GSDMD pores initiate ninjurin 1-mediated recombinant adeno-associated virus
plasma membrane rupture, resulting in the release of larger cellular damage-associated molecular (AAV) to the lung; these are used as
mouse models of innate and adaptive
patterns (DAMPs) that can contribute to inflammation [13]. NLRP3 inflammasome activators are
immune responses to SARS-CoV-2
diverse but appear to promote ion fluxes across cellular membranes, and these are sensed by infection in humans.
NLRP3 through mechanisms that are still being characterized [11]. Human skin organoids: 3D tissues
constructed with primary human
keratinocytes and fibroblasts that mimic
NLRP3 can be activated by virus-encoded effectors known as viroporins (Figure 1), which are
the architecture of human skin.
ion channels that play crucial roles in virus entry, replication, and egress [14]. Influenza A virus Infected cell protein 27 (ICP27): an
(IAV)-infected bone marrow-derived macrophages (BMDMs) from C57BL/6 mice secrete IL-1β essential herpes simplex virus protein
that is reduced in Nlrp3−/− compared to wild-type (WT) BMDMs, implicating the NLRP3 inflamma- that is responsible for host
transcriptional regulation.
some in this innate immune response [15]. Mutational analysis demonstrated that the ion trans-
Inflammasome: a class of innate
port function of the IAV M2 viroporin protein, as well as its localization and ability to acidify the immune proteins that form multimeric
Golgi apparatus are required for inflammasome activation in BMDMs [15]. Further, activation of complexes with ASC and caspases
the NLRP3 inflammasome in lipopolysaccharide-primed mouse BMDMs by encephalomyocardi- upon stimulation to execute pyroptosis.
Necroptosis: caspase-independent
tis virus (EMCV) viroporin 2B is blocked by Ca2+ chelation, indicating that changes in [Ca2+] proinflammatory cell death mechanism
contribute to EMCV 2B-mediated NLRP3 activation [16]. Of note, although IAV M2 protein is mediated by the formation of MLKL-
characterized as a proton transporter [17], transient expression of this protein in HEK293T cells dependent plasma membrane pores.
also increases intracellular [Ca2+] [18], which is interesting because the requirement for Ca2+ Pathogen-associated molecular
patterns (PAMPs): pathogen-
fluxes in M2 protein-mediated NLRP3 activation, to our knowledge, remain to be reported. associated molecules with conserved
motifs that trigger specific PRRs and
NLRP3 may also be triggered in humans by viroporins produced by pathogenic coronaviruses signaling pathways.
Pattern recognition receptors
such as severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 [19–23].
(PRRs): germline-encoded sensors
SARS-CoV produces three viroporins: ORF8, ORF3a, and E, but only expression of the latter that detect invariant microbial products
protein is sufficient to promote NLRP3 inflammasome activation in vitro [19,24]. Transient expression and engage signaling pathways that
of SARS-CoV E in Vero cells increases cytosolic [Ca2+], and mutational analysis suggests that its ion result in transcription or cell-death.
Pattern-triggered immunity (PTI):
channel activity is necessary to trigger reconstituted NLRP3 inflammasome signaling in these cells
immune responses triggered by PRR
[19]. Recombinant SARS-CoV that produces a mutant E protein with disrupted ion channel activity signaling.
replicates as efficiently as WT SARS-CoV in mice, but the former mice show higher survival rates Pyroptosis: a proinflammatory cell
than those infected with WT virus, suggesting a role for viroporin activity in disease severity associ- death mechanism mediated by plasma

ated with SARS-CoV infection [25].

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Inflammasome activation may also play a role in the immunopathology observed in SARS-CoV-2- membrane pore formation via
gasdermin family members.
infected patients with severe coronavirus disease 2019 (COVID-19) [26]. Blood monocytes and SCF E3 ubiquitin ligase complex: a
tissue-resident macrophage from COVID-19 patients show evidence of active NLRP3 family of multisubunit E3 ubiquitin ligase
inflammasomes, such as NLRP3 puncta and ASC specks [20,22], and the NLRP3 inhibitors complexes that are responsible for
glyburide and MCC950 decrease IL-1β cytokine release from SARS-CoV-2-infected human proteasome-dependent degradation of
ubiquitinated proteins.
monocytes in vitro [20,27]. In addition, ASC specks are observed in lung macrophages isolated Self-guarded proteins: cellular
from a humanized mouse model of SARS-CoV-2 infection, and are inhibited by in vivo proteins that have a primary innate
administration of MCC950 [23]. Despite the evidence for NLRP3 activation following SARS- immune defense function which, when
CoV-2 infection, the contribution of viroporins to this process remains unclear. ORF3a and E disrupted by pathogen effectors, acti-
vates a secondary protective immune
homologs are encoded by SARS-CoV-2, whereas ORF8 appears to be deleted in several viral response.
variants [28]. One study reported that SARS-Cov-2 ORF3a-mediated activation of caspase-1 Viroporins: small, hydrophobic,
and IL-1β release in A549 cells were decreased following treatment with MCC950, implicating multifunctional viral proteins that
oligomerize in host membranes to form
NLRP3 in this process [29]. In addition, in a recent preprint that warrants further validation,
pores.
investigators preliminarily reported that coexpression of ORF3a and E promoted IL-1β release
from HEK293T cells that were reconstituted with NLRP3 inflammasome components [30]. This
was associated with increased mitochondrial [Ca2+] and production of mitochondrial reactive
oxygen species (mROS) which, when blocked via MnTBAP treatment, reduced IL-1β release
from these cells [30]. Further, treatment with N-methyl-4-isoleucine-cyclosporin, an inhibitor of the
mitochondrial permeability transition pore, blocked ORF3a/E-induced IL-1β release, implicating this
pore and the cytosolic accumulation of oxidized mitochondrial DNA (Ox-mtDNA) [31,32] in the
activation of NLRP3 – findings that also require further validation [30]. By contrast, another study
reported no activation of NLRP3 in HEK293T cells or THP1 monocytes transfected with constructs
encoding SARS-CoV-2 ORF3a or E protein [21]. Instead, the SARS-CoV-2 nucleocapsid (N) was
sufficient to activate inflammasome signaling by directly interacting with NLRP3 and promoting its
association with ASC, suggesting a viroporin-independent mechanism of NLRP3 activation [21].
These conflicting observations in vitro may reflect differences in the cell types or models used to
study viroporin activity or the requirement for simultaneous expression of SARS-CoV-2 ORF3a
and E in robust NLRP3 activation. Further investigation is therefore necessary to understand the
mechanism by which SARS-CoV-2 activates NLRP3 in vitro and in vivo, as well as the contribution
of ETI to the immunopathology observed during infections with pathogenic coronaviruses.

The shared ability of NLRP3-activating viroporins (i.e., IAV M2, EMCV 2B, and SARS-CoV E)
to either directly or indirectly elevate intracellular [Ca2+] in vitro raises the question of whether
Ca2+-mediated release of Ox-mtDNA is a unifying mechanism of NLRP3 activation triggered by
these effectors. Additional viroporins (e.g., rotavirus NSP4A [33] and JC virus Agnoprotein
[34,35]) also increase cytosolic [Ca2+] but their ability to induce NLRP3 activation has not
been reported. Thus, future studies will be necessary to uncover whether Ca2+-mitochondrial
dynamics [32] play a central role in viroporin-mediated NLRP3 activation. Understanding the
mechanisms underlying viroporin-induced NLRP3 activation can provide important insight into
how these effector proteins are sensed by mammalian cells and contribute to host defense or
immunopathology.

Sensing viral protease activity

Viral proteases are key proteins encoded by most viruses and which play important roles in viral
replication and immune evasion [36]. Recently, several viral proteases have been shown to trigger
NLRP1 and CARD8 inflammasome activation [37–40] (Figure 1 and Box 1). For example, human
rhinovirus 16 (HRV16) infection activates NLRP1-dependent pyroptosis in human keratinocytes
and airway epithelial cells, as demonstrated by ASC oligomerization and IL-1β cytokine release
that is absent in infected NLRP1−/− cells generated by CRISPR-Cas9 [38]. HRV16 3C protease
(3Cpro), which cleaves viral precursor proteins to their mature forms, is sufficient to induce

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Figure 1. Viroporins and viral proteases activate mammalian inflammasome-dependent pyroptosis. (A) Viroporins, which are viral proteins that form ion
channels and modulate host cell homeostasis, can activate NLRP3. Viroporins such as influenza virus (IAV) M2 and encephalomyocarditis virus (EMCV) 2B activate
NLRP3 in murine bone marrow-derived macrophages (BMDMs) [15,16,18]. M2-mediated activation is dependent on ion transport [17]. 2B induces an influx of Ca+2
into the cytosol, which is necessary for NLRP3 activation [17]. SARS-CoV E protein and SARS CoV-2 E/ORF3a induce similar responses in Vero cells and
reconstituted HEK293T models [19]. In reconstituted models, the activation of NLRP3 is associated with increased cytosolic Ca+2, mROS, and possibly Ox-mtDNA
[31,32]. (B) Viral proteases encoded by several picornaviruses (3Cpro), SARS-CoV-2 (NSP5), and HIV-1 cleave human NLRP1 and CARD8 at their N-termini, leading to
the proteasomal degradation of the protein and release of the active UPA-CARD fragment [38]. (A and B) Active NLRP3 and the UPA-CARD fragment of NLRP1
complex with ASC and caspases to induce gasdermin-mediated pyroptosis and IL-1β cleavage [77,78]. Figure created with BioRender.com. Abbreviations: ASC,
apoptosis-associated speck-like protein containing a caspase recruitment domain; CARD8, caspase recruitment domain family member 8; GSDMD, gasdermin D;
NLRP1/3, nucleotide-binding domain, leucine-rich repeat family, pyrin domain-containing proteins 1 and 3; mROS, mitochondrial reactive oxygen species; Ox-mtDNA,
oxidized mitochondrial DNA.

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Box 1. Mechanism of NLRP1 and CARD8 signaling

NLRP1 and CARD8 are unique members of the inflammasome family based on their structures, expression profiles, and
mechanisms of activation. NLRP1 is expressed in both humans and mice, whereas CARD8 is specifically expressed in
humans. NLRP1 is also abundant in barrier epithelia such as keratinocytes [73], whereas CARD8 is preferentially
expressed in blood and lymphoid tissues [74]. Both inflammasomes are activated by viral proteases and talabostat, a
DPP8/9 inhibitor. In addition, NLRP1 is activated by double-stranded RNA and UV light [75,76]. In contrast to NLRP3,
which uses its PYD to recruit ASC, inflammasome seeding by NLRP1 and CARD8 is mediated by a C-terminal caspase
recruitment domain (CARD) which initiates ASC-dependent or -independent recruitment of caspase-1, respectively [41].
Proximal to the CARD is the FIIND region containing adjacent ZU5 and UPA domains that are autoproteolytically cleaved
to produce a C-terminal UPA-CARD fragment [41]. Non-covalent interactions between the UPA-CARD, the N-terminus of
NLRP1, and DPP9 restrain NLRP1 and CARD8 activation [77,78]. The activation of these pathways, irrespective of stimuli,
depends on proteosome-mediated degradation of their N-termini which releases the active UPA-CARD. The N-terminus
of NLRP1 contains PYD, NACHT, and LRR domains which are connected by linker regions [41]. Of note, the linker region
between the PYD and NACHT domains is intrinsically disordered and susceptible to cleavage [79]; this is important
because this linker is hypothesized to be a 'tripwire' [39] which, when targeted by viral proteases, results in inflammasome
activation.

these NLRP1-dependent phenotypes when transiently expressed in HEK293T cells and immor-
talized human keratinocytes [38]. Mutational analysis demonstrated that 3Cpro cleaves human
NLRP1 within the PYD-NACHT linker region between residues Q130 and G131, revealing an
N-terminal glycine that is recognized and ubiquitinated by the cullinZER1/ZYG11b pathway, and
leads to proteasomal degradation of the N-terminus of NLRP1 and inflammasome activation in
HEK293T overexpression models [38]. Another study expanded on this observation by demon-
strating that additional picornaviruses, including coxsackievirus B3, similarly cleave NLRP1 [39].
Both studies also demonstrate that cell types with high expression of NLRP1, such as airway
epithelia and keratinocytes, are particularly susceptible to protease-mediated pyroptosis [38,39].

Recently, the SARS-CoV-2 3CL protease NSP5, that is best known for its ability to process the
viral polyprotein, was shown to activate NLRP1 by targeting its NACHT domain in primary
human lung epithelial cells and A549 cells overexpressing ASC–GFP. In both cell types, SARS-
CoV-2 infections induced NLRP1 cleavage, ASC speck formation, and lactate dehydrogenase
(LDH) release [37]. However, in contrast to chemical-induced NLRP1 signaling (e.g., via the
DPP8/9 inhibitor talabostat), NSP5-mediated NLRP1 activation promoted caspase-3 (CASP3)
cleavage and GSDME pore formation in these cells [37]. This presumably occurs because NSP5
also cleaves GSDMD, likely as an immune evasion strategy to block NLRP1-mediated pyroptosis,
leading to the activation of a parallel signaling pathway, although this remains to be further investi-
gated. Consequently, NLRP1−/− A549 and primary epithelial cells produce more infectious SARS-
CoV-2 than WT cells, suggesting that NLRP1-dependent pyroptosis can restrict virus replication
[37]. However, high amounts of cleaved caspase-3 and GSDME in plasma obtained from patients
with COVID-19 correlated strongly with disease severity [37]. Disease severity and high pyroptotic
markers might also correlate with alterations in PTI, such as the presence of inactivating mutations
in PRR pathways (TBK1, IFNAR1, IRF7) or the presence of anti-IFN antibodies [37]; this in turn may
suggest that effector-triggered NLRP1 activation following SARS-CoV-2 infection might be either
helpful or harmful to the host, depending on the general immune health of the infected individual,
although this remains conjectural.

Similar viral protease-mediated mechanisms have been reported with respect to the activation of
human CARD8 – which shares FIIND and CARD domains with NLRP1 [41]. HIV-1 protease, a
viral protein that cleaves the unprocessed HIV-1 Gag–Pol polyprotein to produce mature virions,
targets CARD8 in infected HEK293T cells, human macrophages, and CD4+ T cells [40]. HIV-1
protease becomes activated during viral budding [42] and CARD8 is therefore not normally
cleaved during HIV-1 infection of human cells [40]. However, when the protease is prematurely
activated in human CD4+ T cells treated with non-nucleoside reverse transcriptase inhibitors

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(NNRTIs), these cells undergo pyroptosis and this is inhibited in CARD8- and CASP1-deficient
cells generated by CRISPR-Cas9, implicating the CARD8 inflammasome in this effector-
triggered response [40]. Further, NNRTI treatment of HIV-1 latently infected CD4+ T cells from
patients reduced viral titers upon latency reversal via stimulation with anti-CD3 and anti-CD28
antibodies, suggesting that CARD8 activation might contribute to eliminating the latent pool of
HIV-1, although this awaits further investigation [40].

Thus, NLRP1 and CARD8 are mediators of ETI that recognize diverse viral proteases and initiate
pyroptosis when activated. Because the primary function of these proteases is not in inflammasome
activation, NLRP1 or CARD8 cleavage by these proteases suggests that these inflammasomes may
have evolved to be 'tripwires' [39] which activate a cell death pathway that restricts viral spread. This,
although true in in vitro models, will require rigorous investigation in vivo.

Sensing inhibition of host cell protein synthesis

Viruses must hijack host cell protein synthesis to promote their own gene expression and inhibit
PTI [43]. Although ETI is activated when bacteria inhibit host cell protein synthesis [5], until
recently it was unclear whether host cells sensed viral manipulation of this process. Human
keratinocytes sense virus-mediated inhibition of host cell protein synthesis by a surveillance
mechanism involving the BCL-2 family members MCL-1 and BCL-xL that inhibit apoptosis
(Figure 2) [44]. These two proteins normally suppress cell death pathways activated by
proapoptotic BCL-2 family members [45], but their simultaneous inactivation upon virus- or
chemical-mediated protein synthesis inhibition induces pyroptosis that is blocked in CRISPR-
Cas9-generated CASP3- or GSDME-deficient keratinocytes [44]. Natural targets of keratino-
cytes, including vesicular stomatitis virus (VSV) and herpes simplex virus 1 (HSV-1), engage this
ETI response [44]. However, pyroptosis in HSV-1-infected keratinocytes is only observed during
infections with a mutant virus that lacks infected cell protein 27 (ICP27), an immediate-early protein
which was previously proposed to block CASP3-dependent apoptosis [46], suggesting that HSV-1
ICP27 can promote keratinocyte survival by inhibiting effector-triggered pyroptosis [44]. Enterovirus
71 (EV71) may also engage this pathway because it was recently reported to promote pyroptosis
in infected HeLa cells, and this was inhibited in CRISPR-Cas9-generated CASP3−/− GSDME−/−
cells [47]; however, whether pyroptosis of these cells is a consequence of host cell protein
synthesis inhibition was not evaluated. In addition, BCL-2 family member expression patterns and
activities can be cell type-specific [48], and it is unclear whether sensing of virus-induced protein
synthesis inhibition is specific to keratinocytes or whether it occurs in other cell types.

Effector-triggered GSDME-mediated pyroptosis in keratinocytes is not intrinsically antiviral because

no increase in VSV yield is observed when pyroptosis is inhibited by BCL-xL overexpression in
these cells [44]. However, IL-1 cytokines released upon pyroptosis restrict VSV production in
dermal fibroblasts in vitro and in human skin organoids, suggesting a multiple cell-type model
of antiviral activity [44,49]. Of note, EV71 replication, as measured by viral transcript abundance,
is not increased in Gsdme−/− mice, but these animals are protected from EV71-induced lethality
following intraperitoneal injection [47]. This finding is interesting because it suggests that GSDME-
mediated pyroptosis might be helpful or harmful to the host depending on the route of infection
and/or the tissue where replication occurs, although this warrants further investigation. Together,
these studies suggest that protein synthesis inhibition following virus infection activates ETI, but it
remains unclear how broadly applicable this response is to different tissues and additional viruses.

Self-guarded mechanisms of antiviral defense

An emerging theme in ETI is the activation of antiviral defense strategies following virus-mediated
inhibition of self-guarded proteins. Self-guarded proteins act as “dead man's switches” which

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Figure 2. Virus-induced inhibition of

host protein synthesis activates
caspase-3-mediated pyroptosis via
GSDME cleavage. The antiapoptotic
proteins MCL-1 and BCL-xL act as guard
proteins that sense host cell protein
synthesis inhibition by pathogens such
as vesicular stomatitis virus (VSV) and
ICP27-deficient herpes simplex virus 1
(HSV-1) in human keratinocytes [44].
The loss or inhibition of these proteins
upon virus-mediated protein synthesis
inhibition triggers mitochondrial outer
membrane permeabilization (MOMP),
caspase-3 activation, and GSDME-
mediated pyroptosis [44,46,47]. The
resulting IL-1 cytokine release can protect
underlying dermal fibroblasts from further
infection [44,49]. Figure created with
BioRender.com. Abbreviations: BCL-xL,
B cell lymphoma-extra large; GSDME,
gasdermin E; IL-1α, interleukin 1α; MCL-
1, myeloid cell leukemia 1.

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activate parallel antiviral pathways when their primary antiviral function is inhibited by viral effectors
such as those encoded by large DNA viruses [50]. Accordingly, the inhibition of self-guarded
proteins can initiate proinflammatory cell death or induce antiviral transcriptional responses, as
discussed in the following section (Figure 3).

Sensing virus-mediated inhibition of caspase-8

One self-guarded mechanism that hosts use to limit virus replication is the detection of virus-
mediated inhibition of caspase-8 downstream of tumor necrosis factor receptor (TNFR) engage-
ment (Box 2). The orthopoxvirus vaccinia virus (VACV) produces the B13 protein which
inactivates several caspases including caspase-8 in mammalian cells [51]. Infection with VACV
sensitizes mouse cells to TNF-induced necroptosis, as demonstrated by increased RIPK3-
dependent cell death in VACV-infected mouse embryonic fibroblasts (MEFs) treated with recom-
binant TNF [52,53]. B13 contributes to this response because cells infected with a B13-deficient
VACV virus are insensitive to TNF-induced cell death [52]. Further, Tnfr2−/− or Ripk3−/− mice fail to
control VACV replication [53–55], highlighting the importance of necroptosis in antiviral defense.
Cowpoxvirus (CPXV) contains a B13 homolog (CrmA) but, unlike VACV, does not sensitize cells
to TNF-mediated necroptosis [56]. This disconnect between VACV- and CPXV-mediated
responses, despite their shared ability to inhibit caspase-8, was recently demonstrated to be
due to a necroptosis inhibitor (vIRD) encoded by CPXV, which promotes degradation of RIPK3
in L929 cells via interactions with the SCF E3 ubiquitin ligase complex [56]. Of note, vIRD is
absent or truncated in VACV strains, possibly because of laboratory adaptation of this virus,
which may explain why VACV-infected cells are sensitive to necroptosis [56]. This is supported

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Figure 3. Virus infection activates self-guarded immune responses in mice and humans. (A) Vaccinia virus (VACV) and cowpox virus (CPXV) inhibit murine
caspase-8 downstream of TNFR signaling, thereby inhibiting apoptosis. However, inhibition of caspase-8 sensitizes cells to necroptosis, resulting in RIPK1/RIPK3-
dependent MLKL activation [84]. Caspase-8 is also inhibited by the herpesvirus-encoded effectors vICA (HCMV and MCMV), ICP6 (HSV-1), and ICP10 (HSV-2) [64].
However, several viruses such as CPXV also use mechanisms to inhibit necroptosis, including the expression of vIRD [56]. (B) HSV-1 induces loss of MORC3 in an
ICP0-dependent manner in human fibroblasts and monocytes. In fibroblasts, the loss of MORC3 is important for enhancing HSV-1 replication [72]; however, in
monocytes it results in IFNB1 transcription and restriction of virus replication [50]. Thus, MORC3 acts a self-guarded protein in a cell type-dependent manner. Figure
created with BioRender.com. Abbreviations: cIAP, cellular inhibitor of apoptosis protein; FADD, Fas-associated via death domain; HCMV, human cytomegalovirus;
HSV-1/2, herpes simplex viruses 1 and 2; ICP0, infected cell protein 0; IFN-β, interferon β; MCMV, murine cytomegalovirus; MLKL, mixed lineage kinase domain-like
protein; MORC, MORC family CW-type zinc finger 3; MRE, MORC regulated element; RIPK1, receptor-interacting serine/threonine protein kinase 1; RIPK3, receptor-
interacting serine/threonine-protein kinase 3; TAK1, transforming growth factor β-activated kinase 1; TNFR, tumor necrosis factor (TNF) receptor; TRADD, TNF
receptor type 1-associated DEATH domain; TRAF2, TNF receptor-associated factor 2.

by evidence that mouse cells infected with recombinant VACV-expressing vIRD are more resis-
tant to necroptosis, and that the virus replicates better than unmodified VACV in C57BL6/J
mice [56]. Conversely, replication in mice of recombinant CPXV lacking vIRD was decreased
relative to WT virus, highlighting the importance of vIRD in promoting virus replication in vivo
[56]. In addition, vIRD orthologs are observed in other orthopoxviruses such as monkeypox
virus (MPXV) and ectromelia virus (ECTV), suggesting that this may be a common strategy
among orthopoxviruses to inhibit necroptosis induced by effector-mediated manipulation of
caspase-8 [56].

Caspase-8 is also inhibited by herpesvirus-encoded effectors, including vICA produced by

human and murine cytomegaloviruses (HCMV and MCMV) [57]. Transiently expressed vICA
binds to the pro-domain of caspase-8 and inhibits its proteolytic activation and apoptosis in

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Box 2. Mechanisms of TNFR-induced cell death

In the absence of infection, TNFR signaling promotes the formation of the multiprotein complex I that contains TRADD,
TRAF2, cIAP1/2, and RIPK1 [80]. cIAP1/2-mediated ubiquitination of RIPK1 promotes proinflammatory and prosurvival
transcriptional responses mediated by TAK1 [81] and downstream activation of NF-κB. When cIAP1/2, TAK1, and/or tran-
scription are inhibited by chemicals or pathogens, non-ubiquitinated RIPK1 engages a FADD/caspase-8/cFLIP complex
(complex IIa) resulting in proteolytic activation of caspase-8. Active caspase-8 directly or indirectly promotes the cleavage
of the executioner caspase-3, GSDMD, and GSDME, leading to non-inflammatory apoptotic or proinflammatory
pyroptotic cell death in a cell type-dependent manner [44,82,83]. Inhibition of caspase-8 sensitizes cells to necroptosis
[84]. When caspase-8 is inhibited, non-ubiquitinated RIPK1 seeds complex IIb by recruiting RIPK3 via RIP homotypic
interacting motifs (RHIM) and mixed linkage kinase domain-like (MLKL) pseudokinase via its interaction with RIPK3. RIPK1
phosphorylates RIPK3, which in turn phosphorylates MLKL leading to its oligomerization and transit to the plasma mem-
brane where it forms pores that promote the release of IL-1α and DAMPs [85]. Thus, caspase-8 is a self-guarded protein
that triggers necroptosis when its primary function (i.e., activating apoptotic caspases) in complex I is inhibited.

human cell lines [58,59]. Consequently, vICA-deficient MCMV replicates poorly in vitro and in
mice, and this defect is reversed in Casp8−/− cells or in the presence of caspase inhibitors [60].
Despite their ability to inhibit caspase-8, HCMV- and MCMV-infected cells are not sensitive to
TNF-mediated necroptosis [61]. This is because, like CPXV, they encode proteins that block
necroptosis downstream of caspase-8 inhibition. Specifically, MCMV produces vIRA, a RHIM-
containing protein that disrupts RIPK1–RIPK3 complex formation in mouse cells [62]. By
contrast, HCMV does not encode vIRA, and the Towne strain inhibits necroptosis through an
undefined mechanism downstream of MLKL phosphorylation in human fibroblasts [61]. Recent
work has identified two inositol phosphate (IP) kinases IPMK and ITPK1 as essential regulators
of necroptosis that act downstream of MLKL phosphorylation [63]; it is tempting to speculate
that HCMV might modulate the activity of these kinases or intracellular phosphatase pools to
inhibit MLKL pore formation, although this remains to be investigated.

HSV-1 and HSV-2 also produce caspase-8 inhibitors. In contrast to MCMV described previously,
which produces individual proteins that target caspase-8 and subsequent necroptosis, HSV-1/2
inhibit caspase-8 and effector-triggered necroptosis in human HT-29 cells via single viral proteins –
ICP6 (HSV-1) and ICP10 (HSV-2) [64]. These proteins constitute the large subunits of the viral
ribonucleotide reductase but have novel N-terminal extensions containing a RHIM motif which
are not observed in other ribonucleotide reductases [64–67]. ICP6 coimmunoprecipitates with
caspase-8 in transfected cells, thus inhibiting its catalytic activity while simultaneously blocking
RHIM-mediated interactions between RIPK1 and RIPK3 [64]. Moreover, a HSV-1 mutant produc-
ing a RHIM-deficient ICP6 protein is sensitive to TNFR-dependent necroptosis [64]; however, a
necroptosis-blocking mutant that does not inhibit caspase-8 has not been successfully generated
because caspase-8 binding by ICP6 appears to be required for the ability of ICP6 to inhibit both
apoptosis and necroptosis [64]. Consequently, recombinant HSV-1 strains lacking ICP6
or producing a mutant ICP6 protein are unable to inhibit capase-8 and are restricted in their
replication following chemical activation of either complex IIa or IIb in HT-29 cells [64] or in mice,
respectively [68]; this suggests that ICP6 plays an important role in controlling cell death to promote
virus replication. However, caution should be taken when interpreting results related to the role of
these cell death pathways in host defense and pathogenesis in in vivo mouse HSV-1 infections
because ICP6 activates, rather than inhibits, necroptosis in mouse cells [66,67].

Additional viruses encode proteins that inhibit caspase-8 (e.g., human papillomavirus E6 and
adenovirus E3 14.7), but it remains unclear whether these viruses trigger the self-guarded
response initiated by caspase-8 inhibition [69,70]. In addition, not all cell types are competent
for necroptosis, either because they do not normally express some complex IIb components or
because their expression can be influenced by in vitro culture conditions [61]. It therefore remains

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Trends in Immunology

to be seen whether additional viral gene products that inhibit TNFR-induced apoptosis at the level Outstanding questions
of caspase-8 also initiate effector-triggered necroptosis in competent cell types. Additional evi- Are additional pathways involved
dence would further consolidate this effector-triggered response as an important host defense in surveillance of mammalian cell
homeostasis by guard proteins that
strategy against virus infections.
engage ETI when perturbed by
virus infection?
Sensing loss of the intrinsic resistance protein MORC3
Recently, a cell death-independent mechanism of self-guarding was described through studies Viruses commonly subvert intrinsic
innate immune responses that directly
of HSV-1 interactions with human monocytes. Specifically, HSV-1 infection of human monocytes
limit virus replication (e.g., MORC3).
induces a type I IFN response that is independent of classic PRR signaling components such as Do host cells sense the inhibition of
STING and IRF3 [50]. HSV-1-mediated IFNB1 transcription is reduced in monocytes infected the primary functions of other intrinsic
with a recombinant HSV-1 mutant lacking ICP0, a viral E3 ubiquitin ligase that promotes the deg- resistance mechanisms, and do they
engage alternative antiviral pathways?
radation of antiviral defense proteins [50,71]. Further, ICP0 production via a doxycycline-inducible
lentivirus is sufficient to induce IFNB1 expression in these cells, and this is blocked when the E3 Are there other examples of effector-
ubiquitin ligase activity of ICP0 is inhibited by deletion of the proteins RING-finger domain [50]. triggered transcriptional responses
This implicates the effector activities of ICP0, rather than the production of a viral PAMP, in gen- that take place during virus infection
of mammalian cells?
erating the IFN response in infected monocytes. Expression of ICP0 induces degradation of the
cellular nuclear domain 10 (ND10) component MORC3, and loss of MORC3 by CRISPR-Cas9 Does effector-triggered inflammasome
is sufficient to induce IFNB1 in monocytes [50]. Although MORC3 depletion by shRNA activation contribute to the control of
virus infections in vivo?
enhanced ICP0-deficient HSV-1 replication in primary human fibroblasts [72], its loss by
CRISPR-Cas9 in monocytes repressed WT HSV-1 replication via type I IFN receptor signaling
[50]. ATAC (assay for transposase-accessible chromatin)-seq was used to demonstrate that a
region upstream of the IFNB1 gene (the MRE) becomes accessible in MORC3−/− monocytes,
but how MORC3 represses the accessibility of this region remains unclear. Further, although
MORC3 is targeted for degradation in other cell types infected with HSV-1, such as human
fibroblasts, IFNB1 expression in the absence of MORC3 is only observed in monocytes, which
is interesting because it remains to be determined what facilitates the cell type-specific activation
of this self-guarded response. Thus, this study identified MORC3 as a self-guarded protein
which, when targeted for degradation by a viral E3 ubiquitin ligase, can trigger an antiviral tran-
scriptional response and restrict virus replication.

Concluding remarks
ETI is an emerging component in the repertoire of host defense strategies used during viral
encounters. As obligate intracellular pathogens, the replication and spread of viruses are linked
to their ability to manipulate host cell pathways, inactivate PTI, and inhibit intrinsic defense
mechanisms. As such, diverse viruses subvert similar cellular processes, raising the question of
whether additional cellular pathways are actively surveyed for their manipulation by viral effectors
(see Outstanding questions). Virus-induced ETI may not be immediately apparent, particularly
following infections with viruses with large coding capacity (e.g., herpesviruses and poxviruses)
because of their propensity to encode multiple inhibitors that block primary and alternative host
defense mechanisms. In addition, the role of ETI in antiviral defense may also depend on the
cell types where these responses are activated. Further, although inflammatory cell death has
been the primary consequence of virus-induced ETI, a recent example of a viral effector that trig-
gers an antiviral transcriptional response broadens possible outcomes to be examined.

Several of the studies highlighted here report a role for ETI in limiting virus replication, whereas
others have demonstrated that ETI can induce immunopathology and decrease host survival
during viral infection. ETI is commonly engaged during infections with highly pathogenic viruses
that robustly inhibit PTI, and ETI may therefore represent a final effort to limit virus replication –
with the caveat that its downstream consequences (e.g., inflammation) may be detrimental to
the host if not properly regulated, or when activated inappropriately. Further studies on the

Trends in Immunology, December 2022, Vol. 43, No. 12 1015


interplay between host and virus with regard to sensing virus-encoded effectors would be impor-
tant to determine the true impact of ETI on virus infection and human disease.
Acknowledgments
M.H.O. is supported by a Smith Family Award for Excellence in Biomedical Research from the Richard and Susan Smith
Family Foundation, Newton, MA, USA.
Declaration of interests
The authors declare no conflicts of interest.
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