CARD8 is an inflammasome sensor for HIV-1 protease activity

Eradicating the last vestiges of HIV After treatment with antiretroviral therapy, HIV-1 wild-type and escape variants can persist in a latent form, especially within CD4+ T cells, hindering efforts to eradicate the virus. Q. Wang et al. found that human CARD8, a member of the caspase recruitment domain (CARD)–containing family of innate immune sensors, is activatable by direct proteolysis of its N-terminus by HIV-1 protease. This cleavage should result in the programmed cell death of infected cells, but HIV-1 protease remains inactive and undetected as a subunit of the unprocessed Gag-Pol polyprotein. However, when infected cells were treated with non-nucleoside reverse transcriptase inhibitors, intracellular Gag-Pol dimerization was enhanced, resulting in CARD8-mediated caspase activation and pyroptosis. Targeting this pathway may be a promising way to eliminate residual HIV-1 in patients. Science, this issue p. eabe1707 Caspase recruitment domain–containing protein 8 is an innate immune sensor of HIV-1 protease function. INTRODUCTION HIV-1 has high mutation rates and exists as mutant swarms within the host. The adaptive immune system recognizes cognate epitopes based on amino acid sequences and associated conformations that are subject to mutation. The within-host diversity of HIV-1 allows rapid selection of antibody and T cell escape variants, leading to viral persistence. In patients who receive antiretroviral therapy (ART), HIV-1 persists in a latent form primarily in quiescent CD4+ T cells. Immune escape variants achieved in the latent viral reservoirs present one of the major obstacles to HIV-1 eradication. Approaches to targeting immutable viral components such as the functions of essential viral proteins are needed to clear HIV-1 infection. RATIONALE The host immune system uses germline-encoded pattern recognition receptors (PRRs) to detect microbial products. A set of intracellular PRRs characterized by the presence of a caspase recruitment domain or a pyrin domain co-oligomerize with pro–caspase-1 (pro-CASP1) to form high-molecular-weight inflammasome complexes upon sensing of their cognate ligands. Inflammasome assembly in response to cytoplasmic microbial or danger signals leads to CASP1 activation and pyroptosis, an inflammatory form of programmed cell death. In the present study, we aimed to identify the inflammasome sensor(s) that recognizes intracellular HIV-1 activity. Caspase recruitment domain–containing protein 8 (CARD8) has been implicated in inflammasome activation and pyroptosis of CD4+ T cells and macrophages. A key question is whether CARD8 is an inflammasome sensor and, if so, which pathogens physiologically activate it. RESULTS The C terminus of CARD8 contains a “function-to-find” domain (FIIND) followed by a CARD domain. CARD8 undergoes autoproteolytic processing in the FIIND domain at position 296, generating the N-terminal ZU5 and C-terminal UPA-CARD fragments that remain associated noncovalently. Human CARD8 shares structural similarity with the C terminus of human and murine nucleotide-binding domain leucine-rich repeat pyrin domain–containing 1 (NLRP1), which can be cleaved and activated by microbial proteases. We found that CARD8 could be activated by direct proteolysis of its N terminus by HIV-1 protease. N-terminal cleavage of CARD8 by HIV-1 protease creates an unstable neo–N terminus, which is targeted for proteasome degradation. Because of the break in the polypeptide chain by FIIND autoprocessing, the bioactive UPA-CARD subunit is liberated from the proteasome complex and initiates CASP1-dependent inflammasome assembly. In HIV-1–infected cells, CARD8 cannot detect the virus because the viral protease remains inactive as a subunit of unprocessed Gag–Pol polyprotein. Unexpectedly, some HIV-1–specific non-nucleoside reverse transcriptase inhibitors (NNRTIs) can trigger CARD8 sensing because they bind to HIV-1 Pol and enhance intracellular Gag–Pol polyprotein dimerization. This, in turn, causes premature viral protease activation. Treating HIV-1–infected macrophages and CD4+ T cells with NNRTIs leads to CARD8-mediated caspase 1 activation and pyroptotic cell death. The viral protease activity against CARD8 is well conserved across major HIV-1 subtypes. To test whether strategies involving targeted activation of the CARD8 inflammasome could be used for the clearance of latent HIV-1, we obtained blood CD4+ T cells from patients undergoing suppressive ART to measure the size of viral reservoirs. With NNRTI treatment, we showed that the induction of the CARD8 inflammasome activation led to rapid clearance of latent HIV-1 in patient CD4+ T cells after virus reactivation. CONCLUSION In this study, we report that the CARD8 inflammasome senses HIV-1 protease activity. NNRTIs clear HIV-1–infected cells by inducing viral protease–mediated CARD8 activation. Our study suggests that enhancement of intracellular viral protease activity may be an effective strategy to clear residual HIV-1 in patients. Application of NNRTIs in HIV-1 cure strategies should facilitate the elimination of residual infected cells after the reversal of viral latency. In addition, the inclusion of NNRTIs in the initial ARV regimen may partially reduce the seeding of latent HIV-1 reservoirs. This work reveals a mechanism of innate sensing of HIV-1 infection that has immediate implications for HIV-1 cure strategies. Activation of the CARD8 inflammasome by HIV-1 protease. (1) CARD8 autoproteolytic cleavage. (2) Cleavage of the CARD8 N terminus by HIV-1 protease. (3) Proteosome degradation of the neo–N terminus. (4) Release of the bioactive UPA-CARD fragment. (5) CASP1 activation and pyroptosis of HIV-1–infected cells. (Image was created with BioRender.com.) HIV-1 has high mutation rates and exists as mutant swarms within the host. Rapid evolution of HIV-1 allows the virus to outpace the host immune system, leading to viral persistence. Approaches to targeting immutable components are needed to clear HIV-1 infection. Here, we report that the caspase recruitment domain–containing protein 8 (CARD8) inflammasome senses HIV-1 protease activity. HIV-1 can evade CARD8 sensing because its protease remains inactive in infected cells before viral budding. Premature intracellular activation of the viral protease triggered CARD8 inflammasome–mediated pyroptosis of HIV-1–infected cells. This strategy led to the clearance of latent HIV-1 in patient CD4+ T cells after viral reactivation. Thus, our study identifies CARD8 as an inflammasome sensor of HIV-1, which holds promise as a strategy for the clearance of persistent HIV-1 infection.