Mechanistic Study of HIV Gag in sequestering the CARD8 inflammasome

Abstract Despite the enhancement of antiretroviral therapy (ART) that allows people living with HIV (PLWH) to have an undetectable viral load, HIV-1 persists in a small pool of latently infected, resting memory CD4+ T cells. A major challenge to the immune control and clearance of HIV-1 infection is the rapid within-host viral evolution, which allows selection of viral variants that escape from T cell and antibody recognition. Thus, it is impossible to clear HIV infection without targeting “immutable” components of the virus. Unlike the adaptive immune system that recognizes cognate epitopes or their secondary structures, the CARD8 inflammasome senses the essential enzymatic activity of the HIV-1 protease, which is immutable for the virus. In HIV-1-infected cells, the viral protease is expressed as a subunit of the viral Gag-Pol polyprotein and remains functionally inactive prior to viral budding. Some non-nucleoside reverse transcriptase inhibitors (NNRTIs) can promote intracellular Gag-pol dimerization and subsequent premature intracellular activation of the viral protease. NNRTI treatment triggers CARD8 inflammasome activation, which leads to pyroptosis of HIV-infected CD4+ T cells and macrophages. Thus, targeting the CARD8 inflammasome can be a potent and broadly effective strategy for HIV eradication. Recently, we found that HIV-1 Gag/matrix binds to CARD8 and prevents its activation by the viral protease. This interaction reduces NNRTI efficacy in clearing HIV-1-infected cells. Our goal is to develop a complete and atomically detailed mechanism of CARD8 sequestration by HIV-1 Gag/matrix. In this project, we will combine structural, biochemical, and functional approaches to elucidate the molecular details of the interplay between HIV Gag/matrix and the CARD8 inflammasome. The specific aims in this proposal include 1) functional characterization of CARD8 interaction with HIV- 1 Gag and MA, and comparison of CARD8 inhibition by matrix from different HIV-1 subtypes; and 2) structural and biochemical elucidation of HIV-1 Gag- and MA-mediated CARD8 sequestration. Our study can elucidate the molecular mechanisms of HIV and CARD8 interaction and provide critical insights to CARD8-based drug development.