Anti-capsid nanobodies function as HIV-1 capsid inhibitors by intracellular capsid degradation.
Antiretroviral therapy (ART) strategies against HIV-1 have been successful in suppressing HIV-1 replication and preventing disease progression. However, drug-resistance development, side-effects and life-long adherence of current drugs have driven the development of novel inhibitors against HIV-1. Inhibition of capsid production by targeting the highly conserved capsid protein (CA) has shown to be promising in blocking viral replication. Here, we developed a capsid-targeting biological composed of a single-domain Llama VHH that binds CA, fused to human IgG1 Fc to activate TRIM21-mediated degradation. This anti-capsid biological (aCA-Fc) efficiently bound CA. When expressed intracellularly aCA-Fc completely blocked HIV-1 production by degrading the capsid precursor Gag. Moreover, transfection of purified aCA-Fc during HIV-1 infection also resulted in degradation of Gag. aCA-Fc inhibited HIV-1 replication to similar extent as clinically approved capsid inhibitor lenacapavir (LEN). Interestingly, aCA-Fc didn't interfere with viral entry and reverse transcription, but eliminated newly produced HIV-1 Gag via TRIM21-mediated proteasomal degradation, even when added after infection. Gag production was restored by proteasome inhibition, introduction of the H433A mutation in the Fc domain and by TRIM21 knockdown using siRNA. These findings suggest that capsid-targeting biologicals, upon effective intracellular delivery, could serve as novel therapeutic strategies for viral suppression through intracellular protein degradation.