The immune system recognizes disease-causing microbes, such as bacteria and viruses, and removes them from the body before they can cause harm. When the immune system first detects these foreign invaders, a multi-part structure known as the inflammasome launches an inflammatory response to help fight the microbes off. Several sensor proteins can activate the inflammasome, including one in mice called NLRP1B. This protein has evolved a specialized site that can be cut by a bacterial toxin. Once cleaved, this region acts like a biological tripwire and sparks NLRP1B into action, allowing the sensor to activate the inflammasome system.

Humans have a similar protein called NLRP1, but it is unclear whether this protein has also evolved a tripwire region that can sense microbial proteins. To answer this question, Tsu, Beierschmitt et al. set out to find whether NLRP1 can be activated by viruses in the Picornaviridae family, which are responsible for diseases like polio, hepatitis A, and the common cold. This revealed that NLRP1 contains a cleavage site for enzymes produced by some, but not all, of the viruses in the picornavirus family. Further experiments confirmed that when a picornavirus enzyme cuts through this region during a viral infection, it triggers NLRP1 to activate the inflammasome and initiate an immune response.

The enzymes from different viruses were also found to cleave human NLRP1 at different sites, and the protein’s susceptibility to cleavage varied between different animal species. For instance, Tsu, Beierschmitt et al. discovered that NLRP1B in mice is also able to sense picornaviruses, and that different enzymes activate and cleave NLRP1B and NLRP1 to varying degrees: this affected how well the two proteins are expected to be able to sense specific viral infections. This variation suggests that there is an ongoing evolutionary arms-race between viral proteins and the immune system: as viral proteins change and new ones emerge, NLRP1 rapidly evolves new tripwire sites that allow it to sense the infection and launch an inflammatory response.

What happens when NLRP1B activates the inflammasome during a viral infection is still an open question. The discovery that mouse NLRP1B shares features with human NLRP1 could allow the development of animal models to study the role of the tripwire in antiviral defenses and the overactive inflammation associated with some viral infections. Understanding the types of viruses that activate the NLRP1 inflammasome, and the outcomes of the resulting immune response, may have implications for future treatments of viral infections.

The ability to sense and respond to pathogens is central to the mammalian immune system. However, immune activation needs to be properly calibrated, as an overactive immune response can at times be as pathogenic as the pathogen itself. To ensure accurate discrimination of self and non-self, innate immune sensors detect broadly conserved microbial molecules such as bacterial flagellin or double-stranded RNA (Janeway, 1989). However, such microbial patterns can be found on harmless and pathogenic microbes alike. More recently, pathogen-specific activities such as toxins or effector enzymes have also been shown to be targets of innate immune recognition (Jones et al., 2016; Mitchell et al., 2019; Vance et al., 2009). Such a system for detection, termed effector-trigged immunity (ETI), has been well-established in plants (Cui et al., 2015; Jones et al., 2016) and is emerging as an important means to allow the immune system to distinguish pathogens from harmless microbes in animals (Fischer et al., 2020; Jones et al., 2016).

Complicating the success of host detection systems, innate immune sensors are under constant selective pressure to adapt due to pathogen evasion or antagonism of immune detection. Such evolutionary dynamics, termed host-pathogen arms races, result from genetic conflicts where both host and pathogen are continually driven to adapt to maintain a fitness advantage. The antagonistic nature of these conflicts can be distinguished via signatures of rapid molecular evolution at the exact sites where host and pathogen interact (Daugherty and Malik, 2012; Meyerson and Sawyer, 2011; Sironi et al., 2015). Consistent with their role as the first line of cellular defense against incoming pathogens, innate immune sensors of both broad molecular patterns as well as specific pathogen-associated effectors have been shown to be engaged in genetic conflicts with pathogens (Cagliani et al., 2014; Chavarría-Smith et al., 2016; Hancks et al., 2015; Tenthorey et al., 2014; Tian et al., 2009).

Inflammasomes are one such group of rapidly evolving cytosolic immune sensor complexes (Broz and Dixit, 2016; Chavarría-Smith et al., 2016; Evavold and Kagan, 2019; Rathinam and Fitzgerald, 2016; Tenthorey et al., 2014; Tian et al., 2009). Upon detection of microbial molecules or pathogen-encoded activities, inflammasome-forming sensor proteins serve as a platform for the recruitment and activation of proinflammatory caspases including caspase-1 (CASP1) through either a pyrin domain (PYD) or a caspase activation and recruitment domain (CARD) (Broz and Dixit, 2016; Rathinam and Fitzgerald, 2016). Active CASP1 mediates the maturation and release of the proinflammatory cytokines interleukin (IL)−1β and IL-18 (Broz and Dixit, 2016; Rathinam et al., 2012). CASP1 also initiates a form of cell death known as pyroptosis (Broz and Dixit, 2016; Rathinam et al., 2012). Together, these outputs provide robust defense against a wide array of eukaryotic, bacterial, and viral pathogens (Broz and Dixit, 2016; Evavold and Kagan, 2019; Rathinam and Fitzgerald, 2016).

The first described inflammasome is scaffolded by the sensor protein NLRP1, a member of the nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing (NLR) superfamily (Martinon et al., 2002; Ting et al., 2008). NLRP1 has an unusual domain architecture, containing a CARD at its C-terminus rather than the N-terminus like all other inflammasome sensor NLRs, and a function-to-find domain (FIIND), which is located between the LRRs and CARD (Ting et al., 2008). The FIIND undergoes a constitutive self-cleavage event, such that NLRP1 exists in its non-activated state as two, noncovalently associated polypeptides (D'Osualdo et al., 2011; Finger et al., 2012; Frew et al., 2012), the N-terminal domains and the C-terminal CARD-containing fragment.

The importance of the unusual domain architecture of NLRP1 for mounting a pathogen-specific inflammasome response has been elucidated over the last several decades (Evavold and Kagan, 2019; Mitchell et al., 2019; Taabazuing et al., 2020). The first hint that NLRP1 does not detect broadly conserved microbial molecules came from the discovery that the Bacillus anthracis Lethal Toxin (LeTx) is required to elicit a protective inflammatory response against B. anthracis infection via one of the mouse NLRP1 homologs, NLRP1B (Boyden and Dietrich, 2006; Greaney et al., 2020; Moayeri et al., 2010; Terra et al., 2010). Paradoxically, inflammasome activation is the result of site-specific cleavage in the N-terminus of mouse NLRP1B by the Lethal Factor (LF) protease subunit of LeTx, indicating that protease-mediated cleavage of NLRP1 does not disable its function but instead results in its activation (Chavarría-Smith and Vance, 2013; Levinsohn et al., 2012). More recently, the mechanism by which LF-mediated proteolytic cleavage results in direct NLRP1B inflammasome activation has been detailed (Chui et al., 2019; Sandstrom et al., 2019). These studies revealed that proteolysis of mouse NLRP1B by LF results in exposure of a novel N-terminus, which is then targeted for proteasomal degradation by a protein quality control mechanism called the ‘N-end rule’ pathway (Chui et al., 2019; Sandstrom et al., 2019; Wickliffe et al., 2008; Xu et al., 2019). Since the proteasome is a processive protease, it progressively degrades the N-terminal domains of NLRP1B but is disengaged upon arriving at the self-cleavage site within the FIIND domain. Degradation of the N-terminal domains thus releases the bioactive C-terminal CARD-containing fragment of NLRP1B from its non-covalent association with the N-terminal domains, which is sufficient to initiate downstream inflammasome activation (Chui et al., 2019; Sandstrom et al., 2019). By directly coupling NLRP1 inflammasome activation to cleavage by a pathogen-encoded protease, NLRP1 can directly sense and respond to the activity of a pathogen effector. Such a model indicates that the N-terminal domains are not required for NLRP1 activation per se, but rather serve a pathogen-sensing function. Interestingly, the N-terminal ‘linker’ region in mouse NLRP1B that is cleaved by LF is rapidly evolving in rodents, and the analogous linker region is likewise rapidly evolving in primate species (Chavarría-Smith et al., 2016). These data suggest that selection from pathogens has been driving diversification of this protease target region of NLRP1, possibly serving to bait diverse pathogenic proteases into cleaving NLRP1 and activating the inflammasome responses.

Consistent with the rapid evolution in NLRP1 at the site of proteolytic cleavage, LF neither cleaves nor activates human NLRP1 (Mitchell et al., 2019; Moayeri et al., 2012; Taabazuing et al., 2020). Despite this, human NLRP1 can also be activated by proteolysis when a tobacco etch virus (TEV) protease site is engineered into the rapidly evolving linker region of human NLRP1 that is analogous to the site of LF cleavage in mouse NLRP1B (Chavarría-Smith et al., 2016). Thus, like mouse NLRP1B, it has been predicted that human NLRP1 may serve as a ‘tripwire’ sensor for pathogen-encoded proteases (Mitchell et al., 2019).

Here, we investigate the hypothesis that viral proteases cleave and activate human NLRP1. We reasoned that human viruses, many of which encode proteases as necessary enzymes for their replication cycle, may be triggers for NLRP1 activation. To pursue this hypothesis, we focused on viruses in the Picornaviridae family, which encompass a diverse set of human enteric and respiratory pathogens including coxsackieviruses, polioviruses, and rhinoviruses (Zell, 2018). These viruses all translate their genome as a single polyprotein, which is then cleaved into mature proteins in at least six sites in a sequence-specific manner by a virally encoded 3C protease, termed 3C^pro (Laitinen et al., 2016; Solomon et al., 2010; Sun et al., 2016; Zell, 2018). 3C^pro is also known to proteolytically target numerous host factors, many of which are associated with immune modulation (Croft et al., 2018; Huang et al., 2015; Lei et al., 2017; Mukherjee et al., 2011; Qian et al., 2017; Wang et al., 2019; Wang et al., 2012; Wang et al., 2014; Wang et al., 2015; Wen et al., 2019; Xiang et al., 2014; Xiang et al., 2016; Zaragoza et al., 2006). Because 3C^pro are evolutionarily constrained to cleave several specific polyprotein sites and host targets for replicative success, we reasoned that human NLRP1 may have evolved to sense viral infection by mimicking viral polyprotein cleavage sites, leading to NLRP1 cleavage and inflammasome activation. Using an evolution-guided approach, we now show that NLRP1 is cleaved in its rapidly evolving linker region by several 3C^pro from picornaviruses, resulting in inflammasome activation. These results are consistent with the recent discovery that human rhinovirus (HRV) 3C^pro cleaves and activates NLRP1 in airway epithelia (Robinson et al., 2020). We also find that variation in the cleavage site among primates, and even within the human population, leads to changes in cleavage susceptibility and inflammasome activation. Interestingly, we observe that proteases from multiple genera of viruses cleave and activate human NLRP1 and mouse NLRP1B at different sites, supporting a role for an evolutionary conflict between viral proteases and NLRP1. Taken together, our work highlights the role of NLRP1 in sensing and responding to diverse viral proteases by evolving cleavage motifs that mimic natural sites of proteolytic cleavage in the viral polyprotein.

Pathogens and their hosts are locked in a continual evolutionary conflict in which each side is attempting to exploit the others’ weakness. One particularly successful strategy that pathogens have adopted is to exploit host processes that are highly constrained, leaving the host little room to evolutionarily adapt to overcome the pathogen. For instance, molecular mimicry of host proteins is commonly deployed by pathogens to antagonize host defenses, as it limits the evolutionary options for the host to counter-evolve (Elde and Malik, 2009). Beyond mimicry of entire proteins or protein domains, pathogens can also mimic so-called ‘short linear motifs’ (SLIMs) through evolution of only a small number of amino acids to hijack highly conserved host processes such as post-translational modifications or binding by small protein domains (Chemes et al., 2015; Hagai et al., 2014). Although these strategies are generally described as taking advantage of host evolutionary constraint, pathogens also have potential weak points of evolutionary constraint. In particular, proteases from positive-sense RNA viruses, such as picornaviruses, need to specifically cleave numerous sites within the viral polyprotein in order to reproduce. Thus, changing protease specificity requires concomitant changes to several independent cleavage sites, which is difficult to accomplish in a single evolutionary step. On top of that, protease cleavage motifs often only span a small number of amino acids (Schechter and Berger, 1967), potentially facilitating the independent evolution of these SLIMs in host proteins.

Here, we show that the inflammasome protein, NLRP1, serves as a sensor for diverse proteases from the Picornaviridae family of human pathogens by mimicking the highly conserved protease cleavage sites found within the viral polyproteins. By exploiting a constrained feature of viral evolution and tying it to a pro-inflammatory immune response, such a system allows the immune system to recognize and respond to a wide range of viral proteases expressed in the host cytoplasm. NLRP1 represents one of the few known cases of mammalian ETI (Cui et al., 2015; Fischer et al., 2020; Jones et al., 2016), where pathogen-mediated cleavage of NLRP1 promotes its activation. By holding the small C-terminal CARD-containing fragment in a non-covalent association with the larger N-terminal fragment, the majority of the protein can serve as a sensor for pathogen-encoded effectors (Mitchell et al., 2019; Taabazuing et al., 2020). This presents an opportunity to allow NLRP1 to evolve to be recognized by pathogenic effectors, ultimately leading to degradation of the N-terminal fragment. Indeed, mouse NLRP1B has been shown to be specifically cleaved by the protease-containing secreted effector from B. anthracis (LF) as well as being ubiquitylated by an E3-ubiquitin ligase from S. flexneri (IpaH7.8) (Sandstrom et al., 2019). While these two examples provide evidence that the mouse NLRP1B inflammasome operates by a ‘functional degradation’ model, a direct pathogen-encoded activator of human NLRP1 had remained elusive. We now show, using an evolution-guided approach, that proteases from diverse picornaviruses, including human pathogens such as coxsackievirus B3 (CVB3), human rhinovirus A (HRV), enterovirus D68 (EV68) and poliovirus 1 (PV1) and rosavirus A2 (Rosa2), specifically cleave several independently evolved sites in human NLRP1, leading to activation of the NLRP1 inflammasome and release of pro-inflammatory cytokines such as IL-1β. Together with recent findings (Robinson et al., 2020), our work has thus identified proteases from a diverse range of picornaviruses as pathogen-encoded activators of human NLRP1.

We previously speculated that the unique domain architecture of NLRP1 would allow the N-terminal linker of human NLRP1 to freely evolve to be recognized by pathogenic effectors. Indeed, by harvesting publicly available enterovirus polyprotein sequences for known 3C^pro cleavage sites, we created a 3C^pro cleavage motif that was used to successfully predict the site of enterovirus 3C^pro cleavage at position 130–131 within the rapidly-evolving linker NLRP1. Additionally, our finding that numerous enteroviruses also cleave at the Q130-G131 site and activate pro-inflammatory cytokine release suggests that human NLRP1 serves as a general enteroviral protease sensor by encoding a polyprotein cleavage site mimic. Our phylogenetic assessment of the Q130-G131 3C^pro cleavage site in NLRP1 suggests that NLRP1 sensing of enteroviruses at this specific site is an innovation in the primate lineage, and is largely absent in all other mammalian lineages with exception of a possible independent acquisition by members within the Caprinae subfamily of mammals (e.g. goats, sheep) (Figure 3—figure supplement 1). Interestingly, even within the primate lineage and a small fraction of the human population, some primate orthologs and human variants are cleavage-resistant and therefore do not activate the inflammasome upon cytoplasmic expression of 3C^pro. Such data may hint at three different possible explanations for these changes. First, evolutionary drift in the absence of pressure from pathogenic enteroviruses may account for loss of enterovirus 3C^pro responsiveness in these genes. Second, selection to sense another viral protease may shape the same region of the linker. Finally, while the ETI model of NLRP1 suggests that enteroviral cleavage of NLRP1 has evolved to activate a beneficial immune response in certain contexts, the effects of NLRP1 overactivation may be detrimental in other contexts. In human skin keratinocytes, where NLRP1 is regarded as the key inflammasome, all components of the NLRP1 inflammasome are basally expressed and thus poised to elicit an inflammatory response (Zhong et al., 2016). Here, germline mutations in NLRP1 that result in overactivation can cause growth of warts in the upper airway in a condition known as recurrent respiratory papillomatosis (JRRP) (Drutman et al., 2019) and an increase in skin cancer susceptibility and skin disorders such as multiple self-healing palmoplantar carcinoma (MSPC), familial keratosis lichenoides chronica (FKLC) and auto-inflammation with arthritis and dyskeratosis (AIADK) (Grandemange et al., 2017; Herlin et al., 2020; Soler et al., 2013; Zhong et al., 2016; Zhong et al., 2018). Additional recent work has indicated that dsRNA can also activate the NLRP1 inflammasome in human keratinocytes (Bauernfried et al., 2020), adding to the role that NLRP1 may play in the inflammatory response. Beyond the skin, NLRP1 is also basally expressed in tissues such as the gut and brain (D'Osualdo et al., 2015; Kaushal et al., 2015; Kummer et al., 2007), which are sites of picornavirus replication where overactivation upon infection may result in immunopathology. Further in vivo studies will help determine the role of NLRP1 in antiviral immunity and/or immunopathology during viral infection. Facilitating these studies, our discovery that 3C^pro from HRV potently cleaves and activates NLRP1B from B6 but not 129 mice suggests that rhinovirus infection of B6 mice may be a good model for studying the in vivo consequences of viral-mediated NLRP1 inflammasome activation.

Intriguingly, 3C^pros from nearly every genus of human-infecting picornavirus can cleave NLRP1 somewhere in the rapidly evolving linker region between the PYD and NLR domain, although only enteroviruses cleave at the specific site between position 130 and 131. These data suggest that this extended linker, which we previously found showed widespread signatures of positive selection (Chavarría-Smith et al., 2016), may be convergently evolving to mimic cleavage sites from a diverse range of viruses at multiple independent sites. Supporting that model, we observe a similar phenomenon in mouse NLRP1B, where multiple viral proteases cleave at different sites within NLRP1 in a strain-specific manner. These data highlight the important functional differences in cleavage specificity between even closely related 3C^pro that are not accounted for by predictive models. Further studies will be required to understand the precise relationships between sites within NLRP1 and individual protease specificity. Intriguingly, not all these cleavage events lead to inflammasome activation in the same way that enteroviral cleavage does, and we find evidence for antagonism of NLRP1 activation by some 3C^pros, suggesting that additional activities of 3C^pro may be the next step in the arms race, serving to prevent inflammasome activation even after the tripwire has been tripped.

Taken together, our work suggests that host mimicry of viral polyprotein cleavage motifs could be an important evolutionary strategy in the ongoing arms race between host and viruses. Indeed, one explanation for the somewhat surprising observation that the specificity of viral proteases changes at all within a viral family such as the picornaviruses is that there is evolutionary pressure from the host to evolve cleavage sites and protease specificity. Prior work has highlighted the roles that viral proteases can play in antagonizing host immune factors and driving host evolution to avoid being cleaved (Patel et al., 2012; Stabell et al., 2018). In that case, the viral proteases would evolve to antagonize new factors while maintaining polyprotein cleavage. However, mimicry coupled with cleavage-activating immunity as seen with NLRP1 could be an even stronger pressure to shape the protease specificity. By turning the tables, these host processes may drive the type of functional diversification of viral protease specificity that we observe in order to avoid cleaving NLRP1 and other similar ETI factors. We expect that this work may lead to the discovery that such an evolutionary strategy may be more broadly deployed at other sites of host-pathogen conflicts.

All data generated or analyzed during this study are included in the manuscript and supporting files. Sources of sequence information used for figures and figure supplements have been provided. The ViPR database was used to collect enteroviral polyprotein sequences using the Picornaviridae-specific Gene/Protein search tool (https://www.viprbrc.org/brc/vipr_protein_search.spg?method=ShowCleanSearch&decorator=picorna), selecting protein sequences from all enteroviruses with filters for complete genome to include "completely genome only" and a search type to "include Polyproteins in Results" with the Gene Product Name of "polyprotein". Using the advanced options, options for a minimum CDS length of "6000" with "remove duplicate sequences" were selected. The collection of sequences used in this analysis are listed in Supplementary files 1 and 2. The NCBI protein database (https://www.ncbi.nlm.nih.gov/protein) was used to collect sequences for human (NP_127497.1), mouse NLRP1B allele 129 (AAZ40510.1), mouse NLRP1B allele B6 (XM_017314698.2), other mammalian NLRP1 sequences (Supplementary file 7), picornaviral 3C protease sequences (Supplementary file 7), and NCBI RefSeq enterovirus polyprotein sequences. The NCBI RefSeq enterovirus polyprotein sequences were collected from the NCBI protein database using the search phrase "Enterovirus[Organism] AND srcdb_refseq[PROP] NOT cellular organisms[ORGN]" and filtering by sequence length "2000 to 4000" and release date "to 2018/04/31". Human non-synonymous allele counts for NLRP1 (Figure 4C) were collected using gnomAD (https://gnomad.broadinstitute.org/) v2.1.1 with the search term "NLRP1".

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Acceptance summary:

Bacterial proteases activate the NLRP1B inflammasome in mice, but pathogen-derived activators of the human NLRP1 inflammasome are less well characterized. Here, the authors demonstrate that certain picornaviruses, via their 3C protease, trigger host immune responses by activating human NLRP1, thereby revealing a previously unappreciated sensing mechanism for virally-encoded proteases. Genetic approaches suggest this mechanism has adapted throughout primate evolution as a way to give the host an advantage with respect to its ongoing genetic conflict with the virus.

Decision letter after peer review:

Thank you for submitting your article "Diverse viral proteases activate the NLRP1 inflammasome" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Carla Rothlin as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

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Summary:

The anthrax lethal factor protease activates the NLRP1B inflammasome in mice, but pathogen-derived activators of the human NLRP1 inflammasome have not yet been identified. Here, the authors demonstrate that expression of picornavirus 3C protease activates human NLRP1, implicating a sensing mechanism for pathogen-encoded proteases. Computational and functional evolutionary approaches suggest this mechanism has adapted in primates in host-specific and virus-specific manners.

Essential revisions:

The reviewers were uniform in their requirement for demonstration of this mechanism during picornavirus infection, as all of the current studies are with ectopically expressed protease. Additional experiments to solidify conclusions are outlined below.

1a) Reviewer #1: Does 3C from an active viral infection cleave NLRP1 in the transfection system? The authors could pick 1 enterovirus and 1 non-enterovirus from Figure 4B and infect cells transfected with the NLRP1 system, then score cleavage, Ilb secretion etc.

1b) Reviewer #2: The biggest weakness with this study is that the authors only showed that ectopically expressed proteases can cleave human NLRP1. It is not clear from this work if a living virus will actually cleave and activate NLRP1. If the authors could confirm that the virus itself cleaves and activates NLRP1, it would significantly increase the impact of this work.

1c) Reviewer #3: The manuscript is lacking the biological relevance that would come from carrying out the cleavage experiments in cells infected by coxsackievirus B3, or one of the other enteroviruses whose 3C proteases cleave NLRP1 in transfected cells. Unlike the transfection assays reported in the manuscript, an infection delivers the viral genomic RNA into the cytoplasm of infected cells where translation of the genome produces a polyprotein that is then processed by the 3C and 2A protease activities to generate the viral proteins. In the transfection assays described in this manuscript, the plasmids are delivered to the nucleus, where mRNAs are generated and subsequently transported to the cytoplasm where they are translated as capped, monocistronic mRNAs that produce a single mature 3C protease. These major biological differences could produce differing cleavage patterns of NLRP1 based upon cytoplasmic location of viral proteins or differing forms of the different precursors containing viral protease sequences, which may have differing accessibilities to host protein cleavage targets. Also, the levels of 3C protease produced in cells transfected with plasmid expression vectors may be quite different than what is produced during a coxsackievirus B3 infection. Since the levels of 3C protease produced in the cytoplasm would drive the extent of NRPL1 cleavage and inflammasome activation, the authors need to carry out their assays in the context of a bona fide viral infection.

1d) Reviewer #3: For the related enteroviruses being used in this study, the mature 3C protease is not the major source of protease produced during infection by these viruses. Rather it is the 3CD polypeptide precursor that carries out the majority of cleavage events due to its abundance during an infection. Thus, the use of a mature 3C protease in the transfection assays does not mimic an authentic viral infection in terms of which 3C containing enzymes might be capable of cleaving NLRP1 (or not).

2) As there is little if any information on the role of NLRP1 in human enterovirus infection, the authors should demonstrate that enterovirus infection cleaves NLRP1 in relevant primary cell type such as human monocyte-derived macrophages, or primary human dermal fibroblasts or keratinocytes. A comparison to a picornavirus that does not cleave NLRP1 would be most impactful.

3) A nice control would be to show that a proteasome inhibitor blocks inflammasome activation by 3C^pro.

4) In Figure 4D, the authors show that some proteases cleave mouse NLRP1B, but it is not demonstrated if that activates the inflammasome. An IL-1b ELISA or LDH assay should be shown as well.

Essential revisions:

The reviewers were uniform in their requirement for demonstration of this mechanism during picornavirus infection, as all of the current studies are with ectopically expressed protease. Additional experiments to solidify conclusions are outlined below.

1a) Reviewer #1: Does 3C from an active viral infection cleave NLRP1 in the transfection system? The authors could pick 1 enterovirus and 1 non-enterovirus from Figure 4B and infect cells transfected with the NLRP1 system, then score cleavage, Ilb secretion etc.

1b) Reviewer #2: The biggest weakness with this study is that the authors only showed that ectopically expressed proteases can cleave human NLRP1. It is not clear from this work if a living virus will actually cleave and activate NLRP1. If the authors could confirm that the virus itself cleaves and activates NLRP1, it would significantly increase the impact of this work.

1c) Reviewer #3: The manuscript is lacking the biological relevance that would come from carrying out the cleavage experiments in cells infected by coxsackievirus B3, or one of the other enteroviruses whose 3C proteases cleave NLRP1 in transfected cells. Unlike the transfection assays reported in the manuscript, an infection delivers the viral genomic RNA into the cytoplasm of infected cells where translation of the genome produces a polyprotein that is then processed by the 3C and 2A protease activities to generate the viral proteins. In the transfection assays described in this manuscript, the plasmids are delivered to the nucleus, where mRNAs are generated and subsequently transported to the cytoplasm where they are translated as capped, monocistronic mRNAs that produce a single mature 3C protease. These major biological differences could produce differing cleavage patterns of NLRP1 based upon cytoplasmic location of viral proteins or differing forms of the different precursors containing viral protease sequences, which may have differing accessibilities to host protein cleavage targets. Also, the levels of 3C protease produced in cells transfected with plasmid expression vectors may be quite different than what is produced during a coxsackievirus B3 infection. Since the levels of 3C protease produced in the cytoplasm would drive the extent of NRPL1 cleavage and inflammasome activation, the authors need to carry out their assays in the context of a bona fide viral infection.

We have now included three additional sets of experiments that use live virus infections to address the reviewers’ concerns described above. All of these experiments have been done in parallel with two related picornaviruses that we predict should have different abilities to cleave and activate human NLRP1 based on our data with transfected 3C^pros from these viruses. Specifically, based on the activity of the ectopically-expressed proteases, we predicted that infection with CVB3 should result in cleavage and activation of human NLRP1 whereas infection with a related picornavirus, EMCV, should result in neither cleavage nor activation of human NLRP1.

First, in Figure 2E, we show that infection with CVB3 results in cleavage of human NLRP1 that is detectable starting six hours after infection. Cleavage occurs at the same site (residues 130/131) that we observe with transfected protease. Importantly, in Figure 5D, we show that infection by EMCV results in no NLRP1 cleavage. These results from viral infections mirror the 3C^pro cleavage data found in Figure 5B.

Second, in Figure 3C and Figure 5—figure supplement 5, we show that infection with CVB3, but not EMCV, can activate the human NLRP1 inflammasome in transfected HEK293T cells. Virus-mediated NLRP1 activation requires cleavage at site 130/131, recapitulating the viral and host specificity we observe in Figure 5C.

Finally, we identified an immortalized human keratinocyte cell line, HaCaT, to test whether viral infection could activate endogenously expressed NLRP1. We first demonstrated that infection of this cell line with CVB3, but not EMCV, results in IL-1b release. We went on to generate CRISPR knockouts of NLRP1 and CASP1 and showed reduced IL-1b release when these cells were infected with CVB3. These data are now shown in Figure 3D and Figure 5—figure supplement 5.

Together, these data show that viral infection recapitulates the molecular specificity that we describe with ectopically expressed viral proteases, and also demonstrate that viral infection of human keratinocytes results in NLRP1-dependent inflammasome activation.

Also, as we cite in our paper, while we were working on generating these additional experimental data, another paper was published (Robinson et al., 2020) that convergently discovered that 3C^pro can cleave and activate NLRP1. In their paper, they focus primarily on human rhinovirus (HRV) 3C^pro and show, using approaches similar to the ones we took, that infection of airway epithelia with HRV results in NLRP1 inflammasome activation. The concordance of these data with ours highlight the relevance of 3C^pro-mediated cleavage and activation of NLRP1 during picornaviral infection.

1d) Reviewer #3: For the related enteroviruses being used in this study, the mature 3C protease is not the major source of protease produced during infection by these viruses. Rather it is the 3CD polypeptide precursor that carries out the majority of cleavage events due to its abundance during an infection. Thus, the use of a mature 3C protease in the transfection assays does not mimic an authentic viral infection in terms of which 3C containing enzymes might be capable of cleaving NLRP1 (or not).

As described above, we now include viral infection data that indicates that the protease generated during viral infection cleaves and activates NLRP1 with the same specificity that we observe with ectopically expressed 3C^pros. Although we do not formally know whether this activity is performed by mature 3C^pro or by 3CD, we have also made changes in the Results section that acknowledge the role that 3CD plays as a protease during viral infection.

2) As there is little if any information on the role of NLRP1 in human enterovirus infection, the authors should demonstrate that enterovirus infection cleaves NLRP1 in relevant primary cell type such as human monocyte-derived macrophages, or primary human dermal fibroblasts or keratinocytes. A comparison to a picornavirus that does not cleave NLRP1 would be most impactful.

As described above, we have now shown that CVB3 infection of immortalized human keratinocytes (HaCaT cell line) results in NLRP1 inflammasome activation. As per the reviewers’ suggestion, we also tested EMCV and showed that the NLRP1 inflammasome is not activated. Our CVB3 results are consistent with Robinson et al., who show that HRV infection of primary airway epithelia can activate the NLRP1 inflammasome.

3) A nice control would be to show that a proteasome inhibitor blocks inflammasome activation by 3C^pro.

We have now included data in Figure 3—figure supplement 1 that shows three additional controls that support that the 3C^pro-mediated NLRP1 activation we observe is consistent with the previously described mechanism of NLRP1 activation. First, we show that 3C^pro activation of NLRP1 requires auto-processing of the FIIND domain. We also show that treatment with either a chemical inhibitor of the proteasome (MG132) or a chemical inhibitor of the Cullin-RING E3 ubiquitin ligases that are required for the degradation of proteins with a novel N-terminal glycine (MLN4924) reduce the amount of NLRP1 inflammasome activation we see in response to 3C^pro cleavage.

4) In Figure 4D, the authors show that some proteases cleave mouse NLRP1B, but it is not demonstrated if that activates the inflammasome. An IL-1b ELISA or LDH assay should be shown as well.

We were also intrigued by this observation and chose to pursue the cleavage and activation of mouse NLRP1B further. We have included a new Figure 6 that compares cleavage of the 129 allele of NLRP1B with the B6 allele of NLRP1B by a panel of enterovirus 3C^pros. At the reviewer’s request, we have also included parallel activation data in Figure 6B and show, in particular, that cleavage of B6 NLRP1B by rhinovirus 3C^pro results in strong inflammasome activation as measured by IL-1β maturation. Although NLRP1B is not the main focus of the paper, these data add to the overall model that host and virus evolution, even between closely related species or strains, impact 3C^pro activation of NLRP1. We also note that rhinovirus 3C^pro is the first described virus-encoded activator of mouse NLRP1B, and that activation only occurs for B6 NLRP1B. We are therefore optimistic that these observations could be a good starting point for studies that aim to leverage mouse infection models to reveal the in vivo antiviral and immunopathological consequences of viral-mediated NLRP1 inflammasome activation.

Author details

1. Brian V Tsu

   Division of Biological Sciences, University of California San Diego, San Diego, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Christopher Beierschmitt

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0268-8323

2. Christopher Beierschmitt

   Division of Biological Sciences, University of California San Diego, San Diego, United States

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review and editing

   Contributed equally with

   Brian V Tsu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0151-1091

3. Andrew P Ryan

   Division of Biological Sciences, University of California San Diego, San Diego, United States

   Contribution

   Resources, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2630-9837

4. Rimjhim Agarwal

   Division of Immunology & Pathogenesis, University of California Berkeley, Berkeley, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

5. Patrick S Mitchell

   1. Division of Immunology & Pathogenesis, University of California Berkeley, Berkeley, United States
   2. Department of Microbiology, University of Washington, Seattle, United States

   Contribution

   Conceptualization, Resources, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8375-9060

6. Matthew D Daugherty

   Division of Biological Sciences, University of California San Diego, San Diego, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   mddaugherty@ucsd.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4879-9603

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