Background
Neddylation is essential for post-translational regulation of many cellular processes, including transcription and protein degradation. The NEDD8-activating enzyme inhibitor MLN4924 (MLN) can block degradation of HIV restriction factor APOBEC3G (A3G) in newly infected cells. Here we show that inhibiting neddylation via MLN reduces HIV reactivation from latency after treatment with latency reversing agents (LRAs).
Methods
Latently infected cell lines (ACH2, J-Lat 6.3) were treated with MLN prior to, and during, exposure to three mechanistically distinct LRAs: TNFa, PMA/ionomycin, and JQ1. Following LRA treatment +/- MLN, we quantified extracellular p24 via ELISA and assessed intracellular p24 and apoptosis levels via flow cytometry. To assess provirus transcription, HIV gag, TAR, and long LTR transcripts were quantified by RT-qPCR. Infectivity of virions reactivated from replication competent, Vif+, latent HIV was assessed via infection of TZM-bl cells and immunoblots revealed A3G virion incorporation levels. Lastly, tat/rev multiply-spliced RNA (msRNA) was measured in CD4 T cells from ART-suppressed people living with HIV (PLWH) stimulated ex vivo to assess MLN impact on reactivation in a primary cell model of HIV latency.
Results
In ACH2 cells treated with TNFa, 200nM MLN reduced: gag RNA 4.79-fold (p=0.0029, n=3), the percentage of cells expressing intracellular p24 by 44.3% (p=0.0012, n=4), and extracellular p24 production by 2.36-fold (p=0.0102, n=5) relative to DMSO. PMA/ionomycin and JQ1 stimulation of ACH2 yielded parallel results, as did TNFa treatment of J-Lat 6.3. MLN reduced TAR and long LTR transcripts in ACH2 and J-Lat 6.3, indicating decreased initiation of proviral transcription. ACH2 virion infectivity was reduced by 93.3% compared to DMSO (n=3), and Vif-positive virion A3G increased. MLN treatment also induced apoptosis in ACH2 and uninfected CD4 T cells (Fig. A). In CD4 T cells from three ART-suppressed PLWH, 200nM MLN following PMA/ionomycin stimulation reduced tat/rev msRNA by 35%, 67%, and 43% respectively (n=3) (Fig. B).
Conclusions
Neddylation inhibition limits provirus reactivation by reducing HIV transcription, antigen and virus production and infectivity after LRAs. Our work highlights neddylation as a targetable mechanism to modulate proviral reactivation by LRAs, and MLN as a method to diminish reservoir replenishment when combined with other HIV cure strategies during or after ART.