Background
The CARD8 inflammasome is a key driver of innate immune responses to HIV-1. CARD8, containing HIV-1 protease (PR) cleavage sites, responds to PR upon enforced NNRTI-induced dimerization of the viral Gag-Pol polyprotein and premature PR activation. In the absence of antiretrovirals, the apparent lack of CARD8 activation during HIV-1 assembly along with the observation that PR activation is precisely timed during virus release strongly suggests the existence of HIV-1 encoded mechanisms that prevent premature PR activation and CARD8-mediated cell death. In this study, we determined how structural domains of Gag coordinate the timing of PR activity to evade cleavage of CARD8 and facilitate innate immune escape.
Methods
We established an overexpression system in HEK293T cells using transient transfection to assay CARD8 cleavage by wildtype HIV-1 (WT) or mutants with selective inactivation of MA (Δ6-125), CA oligomerization (VK181-2/P224A), NC (zinc-finger deletion, Δ15-49) or p6 (ΔPTAP) domains of Gag. We infected PMA-differentiated THP-1 cells (PMA/THP1) and primary monocyte-derived macrophages (MDM) with WT or Gag-mutant viruses and determined CARD8 inflammasome activation by measuring IL-1β secretion and LDH release. We also infected primary CD4+ T cells (CD4T) with WT or Gag-mutant viruses in the presence or absence of lopinavir (PR inhibitor) to assess efficiency of PR-mediated cell killing.
Results
We found that alteration of HIV-1 assembly site (MA/Δ6-125) or arrest of virus particle release (p6/ΔPTAP) but not RNA binding (NC/Δ15-49) dramatically induced CARD8 inflammasome by de novo assembling Gag in HEK293T, PMA/THP1, and MDM. In the context of MA-mutant infection, CARD8 activation was dependent on CA-CA interactions and MA myristoylation, consistent with a model where CARD8 is cleaved late in the viral life cycle if the assembly process is altered. In CD4T, NC deletion did cause enhanced PR-mediated cell death, but the effect remained less significant than infection with the MA or p6 mutants. Interestingly, replacement of the NC domain with a nonspecific RNA-binding leucine zipper domain (GagZip) was sufficient to abolish PR-mediated activation of CARD8 in CD4T.
Conclusions
This work reveals important structural regulators of PR activation during the assembly of HIV-1 particles, and identifies the MA and p6 domains of Gag as potential targets to pharmaceutically enact killing of myeloid and lymphoid cells productively infected with HIV-1.
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