<i>Ex vivo</i> and <i>in vivo</i> HIV-1 latency reversal by “Mukungulu,” a protein kinase C-activating African medicinal plant extract

New HIV latency-reversing agents (LRAs) are needed that can reactivate and/or eliminate HIV reservoirs. "Mukungulu," prepared from the plant <i>Croton megalobotrys</i> Müll Arg., is traditionally used for HIV/AIDS management in northern Botswana despite an abundance of protein kinase C-activating phorbol esters ("namushens"). Here, we show that Mukungulu is tolerated in mice at up to 12.5 mg/kg while robustly reversing latency in antiretroviral therapy (ART)-suppressed HIV-infected humanized mice at 5 mg/kg. In primary cells from ART-suppressed people living with HIV-1, 1 µg/mL Mukungulu reverses latency at levels similar to or superior to anti-CD3/CD28 positive control, based on HIV gag-p24 protein expression, while the magnitude of HIV reactivation in peripheral blood mononuclear cells corresponds to intact proviral burden in CD4+ T-cells. Bioassay-guided fractionation identifies five namushen phorbol esters that can reactivate HIV, but when combined, they do not match Mukungulu's activity, suggesting the presence of additional enhancing factors. Together, these results identify Mukungulu as a robust natural LRA that is already in use by humans and which may warrant inclusion in future HIV cure and ART-free remission efforts.IMPORTANCECurrent HIV therapies do not act on the latent viral reservoir, which is the major obstacle toward achieving a drug-free HIV remission and/or an HIV cure. "Mukungulu," a bark preparation from <i>Croton megalobotrys</i> Müll Arg., has been documented for its traditional use for HIV/AIDS management in northern Botswana. Here, we show that Mukungulu activates viral reservoirs, a key step toward identifying and potentially eliminating these reservoirs, in both cells from people living with HIV as well as in HIV-infected humanized mice. The majority of this activity is due to the abundance of five phorbol esters ("namushens"). This reverse pharmacology-based approach has therefore identified a potent activator of viral reservoirs that is already traditionally used by humans, which in turn can inform and advance western HIV cure and drug-free remission efforts.