Background:
Despite the success of combination antiretroviral therapy, persistent viral RNA expression in tissue macrophages such as CNS-resident microglia and associated chronic inflammation still remain a major barrier to HIV-1 cure strategies. Our recently published studies reported that cytoplasmic expression of unspliced or intron-containing viral RNA (icRNA) leads to innate immune activation in human macrophages and microglia, though the icRNA sensing mechanism has remained unclear. Inflammasomes are known to play an important role in regulating immune response and secretion of pro-inflammatory cytokines, IL-1β and IL-18, in response to diverse stimuli. In this study, we focused on the role of HIV icRNA expression in triggering innate immune receptors and inducing inflammasome activation.
Methods:
Monocyte-derived macrophages (MDMs), iPSC-derived microglia, and CHME3 microglial cell line were infected with replication-competent HIV-1 or reporter viruses expressing icRNA accessing distinct nucleocytoplasmic RNA export pathways. Infections were performed in the presence or absence of small molecule inhibitors targeting virus replication cycle, or upon knock-down of innate immune sensing pathways. Inflammasome activation was determined by qRT-PCR, immunoblotting, caspase-1 activation (FLICA) and IL-1β ELISA.
Results:
Infection of MDMs and microglia with WT HIV-1, but not M10/Rev-mutant HIV-1 (that lacks cytoplasmic icRNA export) induced NLRP3 expression and IL-1β secretion. Interestingly, MPMV constitutive transport element (CTE)-mediated rescue of icRNA export in Rev/M10 HIV-1 also primed NLRP3 expression and triggered secretion of IL-1β. IL-1β production was independent of viral icRNA sequence, viral protein (Gag-Pol, Vif, Vpr, Vpu, Nef and Env) expression, and was attenuated upon pre-treatment with HIV-1 RT (Efavirenz), integrase (Raltegravir), transcription (Spironolactone) or icRNA export (KPT335) inhibitors, or treatment with a pan-caspase inhibitor (z-VAD). Importantly, knockdown of MAVS, but not STING or abrogation of endosomal TLR nucleic acid sensing pathways, significantly attenuated IL-1β production, confirming that cytosolic HIV-1 icRNA sensing is required for inflammasome activation in macrophages and microglia.
Conclusions:
These results suggest that provirus establishment and persistent cytoplasmic expression of HIV-1 icRNA alone, even in the absence of viral protein expression, is sufficient to activate inflammasomes and might contribute to chronic innate immune activation.