Hydroxylation of the HIV-1 antisense protein promotes immune evasion of HIV-1 via modulation of TBK1.

HIV-1 evades host immunity via a number of virally encoded mediators. Here we show that the HIV-1 antisense protein (ASP) evades host immunity by interfering with the type I interferon (IFN-I) response. Mechanistically, host prolyl hydroxylase 3 (PHD3) hydroxylates ASP at Pro47, enabling the recruitment of the RING finger protein 114 (RNF114) to TANK-binding kinase 1 (TBK1). Subsequently, RNF114 mediates K6-linked ubiquitination of TBK1 at Lys236, suppressing TBK1 activation and the downstream IFN-I response. Conversely, mutation of ASP at Pro47 abolishes this inhibitory effect. In humanized mice, either ASP deletion or treatment with the RNF114 inhibitor EN219 or the PHD3 inhibitor Molidustat enhances antiviral immunity and reduces viral replication. Clinically, RNF114 and PHD3 transcript levels exhibit a positive correlation with viral load in treatment-naive patients. Here we show a distinct HIV-1 immune evasion mechanism involving proline hydroxylation and K6-linked ubiquitination, highlighting therapeutic potential.