Intracellular replication of Pseudomonas aeruginosa in epithelial cells requires suppression of the caspase-4 inflammasome.

Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed that this involves multiple roles played by the type three secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa, comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with the activation of nuclear factor-κB as measured by p65 relocalization and tumor necrosis factor alpha transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the T3SS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa, not fully dependent on bacterially driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa can exhibit an intracellular lifestyle within epithelial cells in vivo and in vitro. The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when P. aeruginosa is intracellular. Results showed that intracellular P. aeruginosa lacking T3SS effectors could elicit rapid cell lysis via the noncanonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wild-type infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular P. aeruginosa, and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.

Stalled but not forgotten: Bacterial exotoxins inhibit translation to activate NLRP1.

In this issue of JEM, companion articles from Pinilla et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20230104) and Robinson et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20230105) demonstrate that ribotoxic stress induced by Pseudomonas aeruginosa and Corynebacterium diphtheriae EEF2-targeting exotoxins leads to NLRP1 inflammasome activation, representing a new mechanism of effector-triggered immunity.

A human-specific motif facilitates CARD8 inflammasome activation after HIV-1 infection.

Inflammasomes are cytosolic innate immune complexes that assemble upon detection of diverse pathogen-associated cues and play a critical role in host defense and inflammatory pathogenesis. Here, we find that the human inflammasome-forming sensor CARD8 senses HIV-1 infection via site-specific cleavage of the CARD8 N-terminus by the HIV protease (HIV-1PR). HIV-1PR cleavage of CARD8 induces pyroptotic cell death and the release of pro-inflammatory cytokines from infected cells, processes regulated by Toll-like receptor stimulation prior to viral infection. In acutely infected cells, CARD8 senses the activity of both de novo translated HIV-1PR and packaged HIV-1PR that is released from the incoming virion. Moreover, our evolutionary analyses reveal that the HIV-1PR cleavage site in human CARD8 arose after the divergence of chimpanzees and humans. Although chimpanzee CARD8 does not recognize proteases from HIV or simian immunodeficiency viruses from chimpanzees (SIVcpz), SIVcpz does cleave human CARD8, suggesting that SIVcpz was poised to activate the human CARD8 inflammasome prior to its cross-species transmission into humans. Our findings suggest a unique role for CARD8 inflammasome activation in response to lentiviral infection of humans.

Host-specific sensing of coronaviruses and picornaviruses by the CARD8 inflammasome.

Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find that the 3CL protease (3CLpro) encoded by diverse coronaviruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CLpro, including 3CLpro-mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8's ability to sense coronavirus 3CLpros and, instead, enables sensing of 3C proteases (3Cpro) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intraspecies variation in inflammasome-mediated viral sensing and immunopathology.

Intracellular replication of Pseudomonas aeruginosa in epithelial cells requires suppression of the caspase-4 inflammasome.

Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed this involves multiple roles played by the type three-secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa, comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with activation of NF-κB as measured by p65 relocalization and TNFα transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the TSSS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa, not fully dependent on bacterially-driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa.

Host specific sensing of coronaviruses and picornaviruses by the CARD8 inflammasome.

Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find that the 3CL protease (3CL pro ) encoded by diverse coronaviruses, including SARS-CoV-2, cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CL pro , including 3CL pro -mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8’s ability to sense coronavirus 3CL pros , and instead enables sensing of 3C proteases (3C pro ) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intra-species variation in inflammasome-mediated viral sensing and immunopathology.

Epithelial Pyroptosis in Host Defense.

Pyroptosis is a lytic form of cell death that is executed by a family of pore-forming proteins called gasdermins (GSDMs). GSDMs are activated upon proteolysis by host proteases including the proinflammatory caspases downstream of inflammasome activation. In myeloid cells, GSDM pore formation serves two primary functions in host defense: the selective release of processed cytokines to initiate inflammatory responses, and cell death, which eliminates a replicative niche of the pathogen. Barrier epithelia also undergo pyroptosis. However, unique mechanisms are required for the removal of pyroptotic epithelial cells to maintain epithelial barrier integrity. In the following review, we discuss the role of epithelial inflammasomes and pyroptosis in host defense against pathogens. We use the well-established role of inflammasomes in intestinal epithelia to highlight principles of epithelial pyroptosis in host defense of barrier tissues, and discuss how these principles might be shared or distinctive across other epithelial sites.

Diverse viral proteases activate the NLRP1 inflammasome.

The NLRP1 inflammasome is a multiprotein complex that is a potent activator of inflammation. Mouse NLRP1B can be activated through proteolytic cleavage by the bacterial Lethal Toxin (LeTx) protease, resulting in degradation of the N-terminal domains of NLRP1B and liberation of the bioactive C-terminal domain, which includes the caspase activation and recruitment domain (CARD). However, natural pathogen-derived effectors that can activate human NLRP1 have remained unknown. Here, we use an evolutionary model to identify several proteases from diverse picornaviruses that cleave human NLRP1 within a rapidly evolving region of the protein, leading to host-specific and virus-specific activation of the NLRP1 inflammasome. Our work demonstrates that NLRP1 acts as a 'tripwire' to recognize the enzymatic function of a wide range of viral proteases and suggests that host mimicry of viral polyprotein cleavage sites can be an evolutionary strategy to activate a robust inflammatory immune response.

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.

NAIP-NLRC4-deficient mice are susceptible to shigellosis.

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease that is a major cause of diarrhea-associated mortality in humans. Mice are highly resistant to Shigella and the lack of a tractable physiological model of shigellosis has impeded our understanding of this important human disease. Here, we propose that the differential susceptibility of mice and humans to Shigella is due to mouse-specific activation of the NAIP-NLRC4 inflammasome. We find that NAIP-NLRC4-deficient mice are highly susceptible to oral Shigella infection and recapitulate the clinical features of human shigellosis. Although inflammasomes are generally thought to promote Shigella pathogenesis, we instead demonstrate that intestinal epithelial cell (IEC)-specific NAIP-NLRC4 activity is sufficient to protect mice from shigellosis. In addition to describing a new mouse model of shigellosis, our results suggest that the lack of an inflammasome response in IECs may help explain the susceptibility of humans to shigellosis.

Molecular characterization of a fungal gasdermin-like protein.

Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulatorof cell death-1 (rcd-1), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassarcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1-1 and rcd-1-2, is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-1-dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible rcd-1-1/rcd-1-2 alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and rcd-1 This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.

Autoinflammatory disease with corneal and mucosal dyskeratosis caused by a novel NLRP1 variant.

OBJECTIVES: Here we investigated a patient with inflammatory corneal intraepithelial dyskeratosis, mucosal inflammation, tooth abnormalities and, eczema to uncover the genetic and immunological basis of the disease. METHODS: On suspicion of an autoinflammatory condition, Sanger sequencing of nucleotide-binding oligomerization domain-like, leucine-rich repeat pyrin domain containing 1 (NLRP1) was performed and combined with an in vitro inflammasome reconstitution assay to measure caspase-1-mediated IL-1ß cleavage, stimulation of patient peripheral blood mononuclear cells (PBMCs) and whole blood to measure IL-1ß, IL-18 production and quantification of apoptosis-associated speck-like protein containing CARD (ASC) speck formation as a measure of inflammasome activation by flow cytometry. RESULTS: Sanger sequencing revealed a novel mutation (c.175G>C, p.A59P; NM_33004.4) in the inflammasome molecule NLRP1 segregating with disease, although with incomplete penetrance, in three generations. We found that patient PBMCs produced increased IL-1ß in response to inflammatory stimuli, as well as increased constitutive levels of IL-18. Moreover, we demonstrate that expression of the identified NLRP1 A59P variant caused spontaneous IL-1ß cleavage to mature IL-1ß. In addition, patient PBMCs responded to NLRP1 stimulation with increased ASC speck formation as a reflection of elevated inflammasome activity. CONCLUSION: We demonstrate that this novel NLRP1 A59P variant caused increased activation of the NLRP1 inflammasome, resulting in constitutively and inducibly elevated IL-1ß and IL-18 synthesis. We suggest the NLRP1 mutation underlies the pathogenesis of this rare autoinflammatory dyskeratotic disease inherited in an autosomal dominant manner with incomplete penetrance in the patient and within the family for several generations.

The NLRP1 inflammasome: new mechanistic insights and unresolved mysteries.

Nucleotide-binding domain, leucine-rich repeat (NLR) proteins constitute a diverse class of innate immune sensors that detect pathogens or stress-associated stimuli in plants and animals. Some NLRs are activated upon direct binding to pathogen-derived ligands. In contrast, we focus here on a vertebrate NLR called NLRP1 that responds to the enzymatic activities of pathogen effectors. We discuss a newly proposed 'functional degradation' mechanism that explains activation and assembly of NLRP1 into an oligomeric complex called an inflammasome. We also discuss how NLRP1 is activated by non-pathogen-associated triggers such as the anti-cancer drug Val-boroPro, or by human disease-associated mutations. Finally, we discuss how research on NLRP1 has led to additional biological insights, including the unexpected discovery of a new CARD8 inflammasome.

Functional degradation: A mechanism of NLRP1 inflammasome activation by diverse pathogen enzymes.

Inflammasomes are multiprotein platforms that initiate innate immunity by recruitment and activation of caspase-1. The NLRP1B inflammasome is activated upon direct cleavage by the anthrax lethal toxin protease. However, the mechanism by which cleavage results in NLRP1B activation is unknown. In this study, we find that cleavage results in proteasome-mediated degradation of the amino-terminal domains of NLRP1B, liberating a carboxyl-terminal fragment that is a potent caspase-1 activator. Proteasome-mediated degradation of NLRP1B is both necessary and sufficient for NLRP1B activation. Consistent with our functional degradation model, we identify IpaH7.8, a Shigella flexneri ubiquitin ligase secreted effector, as an enzyme that induces NLRP1B degradation and activation. Our results provide a unified mechanism for NLRP1B activation by diverse pathogen-encoded enzymatic activities.

Functional and Evolutionary Analyses Identify Proteolysis as a General Mechanism for NLRP1 Inflammasome Activation.

Inflammasomes are cytosolic multi-protein complexes that initiate immune responses to infection by recruiting and activating the Caspase-1 protease. Human NLRP1 was the first protein shown to form an inflammasome, but its physiological mechanism of activation remains unknown. Recently, specific variants of mouse and rat NLRP1 were found to be activated upon N-terminal cleavage by the anthrax lethal factor protease. However, agonists for other NLRP1 variants, including human NLRP1, are not known, and it remains unclear if they are also activated by proteolysis. Here we demonstrate that two mouse NLRP1 paralogs (NLRP1AB6 and NLRP1BB6) are also activated by N-terminal proteolytic cleavage. We also demonstrate that proteolysis within a specific N-terminal linker region is sufficient to activate human NLRP1. Evolutionary analysis of primate NLRP1 shows the linker/cleavage region has evolved under positive selection, indicative of pathogen-induced selective pressure. Collectively, these results identify proteolysis as a general mechanism of NLRP1 inflammasome activation that appears to be contributing to the rapid evolution of NLRP1 in rodents and primates.

Evolutionary Analyses Suggest a Function of MxB Immunity Proteins Beyond Lentivirus Restriction.

Viruses impose diverse and dynamic challenges on host defenses. Diversifying selection of codons and gene copy number variation are two hallmarks of genetic innovation in antiviral genes engaged in host-virus genetic conflicts. The myxovirus resistance (Mx) genes encode interferon-inducible GTPases that constitute a major arm of the cell-autonomous defense against viral infection. Unlike the broad antiviral activity of MxA, primate MxB was recently shown to specifically inhibit lentiviruses including HIV-1. We carried out detailed evolutionary analyses to investigate whether genetic conflict with lentiviruses has shaped MxB evolution in primates. We found strong evidence for diversifying selection in the MxB N-terminal tail, which contains molecular determinants of MxB anti-lentivirus specificity. However, we found no overlap between previously-mapped residues that dictate lentiviral restriction and those that have evolved under diversifying selection. Instead, our findings are consistent with MxB having a long-standing and important role in the interferon response to viral infection against a broader range of pathogens than is currently appreciated. Despite its critical role in host innate immunity, we also uncovered multiple functional losses of MxB during mammalian evolution, either by pseudogenization or by gene conversion from MxA genes. Thus, although the majority of mammalian genomes encode two Mx genes, this apparent stasis masks the dramatic effects that recombination and diversifying selection have played in shaping the evolutionary history of Mx genes. Discrepancies between our study and previous publications highlight the need to account for recombination in analyses of positive selection, as well as the importance of using sequence datasets with appropriate depth of divergence. Our study also illustrates that evolutionary analyses of antiviral gene families are critical towards understanding molecular principles that govern host-virus interactions and species-specific susceptibility to viral infection.

Systematic screen identifies miRNAs that target RAD51 and RAD51D to enhance chemosensitivity.

UNLABELLED: Homologous recombination mediates error-free repair of DNA double-strand breaks (DSB). RAD51 is an essential protein for catalyzing homologous recombination and its recruitment to DSBs is mediated by many factors including RAD51, its paralogs, and breast/ovarian cancer susceptibility gene products BRCA1/2. Deregulation of these factors leads to impaired DNA repair, genomic instability, and cellular sensitivity to chemotherapeutics such as cisplatin and PARP inhibitors. microRNAs (miRNA) are short, noncoding RNAs that posttranscriptionally regulate gene expression; however, the contribution of miRNAs in the regulation of homologous recombination is not well understood. To address this, a library of human miRNA mimics was systematically screened to pinpoint several miRNAs that significantly reduce RAD51 foci formation in response to ionizing radiation in human osteosarcoma cells. Subsequent study focused on two of the strongest candidates, miR-103 and miR-107, as they are frequently deregulated in cancer. Consistent with the inhibition of RAD51 foci formation, miR-103 and miR-107 reduced homology-directed repair and sensitized cells to various DNA-damaging agents, including cisplatin and a PARP inhibitor. Mechanistic analyses revealed that both miR-103 and miR-107 directly target and regulate RAD51 and RAD51D, which is critical for miR-103/107-mediated chemosensitization. Furthermore, endogenous regulation of RAD51D by miR-103/107 was observed in several tumor subtypes. Taken together, these data show that miR-103 and miR-107 overexpression promotes genomic instability and may be used therapeutically to chemosensitize tumors. IMPLICATIONS: These findings demonstrate a role for miR-103 and -107 in regulating DNA damage repair, thereby identifying new players in the progression of cancer and response to chemotherapy.

Circulating microRNA profiling identifies a subset of metastatic prostate cancer patients with evidence of cancer-associated hypoxia.

MicroRNAs (miRNAs) are small (∼22 nucleotide) non-coding RNAs that regulate a myriad of biological processes and are frequently dysregulated in cancer. Cancer-associated microRNAs have been detected in serum and plasma and hold promise as minimally invasive cancer biomarkers, potentially for assessing disease characteristics in patients with metastatic disease that is difficult to biopsy. Here we used miRNA profiling to identify cancer-associated miRNAs that are differentially expressed in sera from patients with metastatic castration resistant prostate cancer (mCRPC) as compared to healthy controls. Of 365 miRNAs profiled, we identified five serum miRNAs (miR-141, miR-200a, miR-200c, miR-210 and miR-375) that were elevated in cases compared to controls across two independent cohorts. One of these, miR-210, is a known transcriptional target of the hypoxia-responsive HIF-1α signaling pathway. Exposure of cultured prostate cancer cells to hypoxia led to induction of miR-210 and its release into the extracellular environment. Moreover, we found that serum miR-210 levels varied widely amongst mCRPC patients undergoing therapy, and correlated with treatment response as assessed by change in PSA. Our results suggest that (i) cancer-associated hypoxia is a frequent, previously under-appreciated characteristic of mCRPC, and (ii) serum miR-210 may be further developed as a predictive biomarker in patients with this distinct disease biology.

An evolutionary perspective on the broad antiviral specificity of MxA.

Germ line encoded antiviral defenses in vertebrate cells tend to be either broadly acting factors that exploit general features of viral replication or effectors with strong pathogen preference by virtue of specific recognition of viral proteins. The Mx GTPases, however, are atypical since they have broad antiviral activity against a wide range of RNA and DNA viruses despite specifically targeting different proteins across virus families. This review presents recent advances in understanding the biochemical properties and evolution of the primate ortholog MxA, and discusses how this information begins to provide molecular insights into the mechanisms behind the intriguing conundrum of how MxA is able to engage a diversity of viral proteins yet elicit antiviral breadth.

Evolution-guided identification of antiviral specificity determinants in the broadly acting interferon-induced innate immunity factor MxA.

MxA is an interferon-induced dynamin-like GTPase with wide-ranging antiviral activity, which hinges upon detection of unique viral structures that differ across virus families. Despite elucidation of its structure, the basis of MxA antiviral specificity remains enigmatic. We used an evolution-guided approach to identify the loop L4 of MxA as a hotspot for recurrent positive selection in primates. Further, we show that single amino acid changes in L4 are necessary and sufficient to explain dramatic differences in species-specific antiviral activity of primate MxA proteins against the orthomyxoviruses Thogoto virus and influenza A virus. Taken together, our findings identify a genetic determinant of MxA target recognition and suggest a model by which MxA achieves antiviral breadth without compromising viral specificity.