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Our previous work demonstrated that CARD8 detects HIV-1 infection by sensing the enzymatic activity of the HIV protease, resulting in CARD8-dependent inflammasome activation (Kulsuptrakul et al., 2023). CARD8 harbors a motif in its N-terminus that functions as a HIV protease substrate mimic, permitting innate immune recognition of HIV-1 protease activity, which when cleaved by HIV protease triggers CARD8 inflammasome activation. Here, we sought to understand CARD8 responses in the context of HIV-1 cell-to-cell transmission via a viral synapse. We observed that cell-to-cell transmission of HIV-1 between infected T cells and primary human monocyte-derived macrophages induces CARD8 inflammasome activation in a manner that is dependent on viral protease activity and largely independent of the NLRP3 inflammasome. Additionally, to further evaluate the viral determinants of CARD8 sensing, we tested a panel of HIV protease inhibitor resistant clones to establish how variation in HIV protease affects CARD8 activation. We identified mutant HIV-1 proteases that differentially cleave and activate CARD8 compared to wildtype HIV-1, thus indicating that natural variation in HIV protease affects not only the cleavage of the viral Gag-Pol polyprotein but also likely impacts innate sensing and inflammation.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
* This version has been updated in response to reviewer feedback at eLife. The revised manuscript includes several additional experiments, including: HIV-1 cell-to-cell transmission data using primary CD4+ T cells and MDMs; a more detailed time course study using our HIV-1 cell-to-cell transmission model to evaluate CARD8 inflammasome activation by incoming viral protease; knockout of NLRP3 in primary MDMs, which further substantiates the importance of CARD8 but not NLRP3 in the inflammasome response to HIV-1; new or repeat experiments encompassing several additional independent donors. Data from these experiments are consistent with our findings in the original manuscript, and extends and further substantiates our observation that the CARD8 inflammasome is activated in response to viral protease activity during HIV-1 cell-to-cell transmission to myeloid cells.