Functional SLC29A3/ENT3 drives autophagic clearance of intracellular viral particles.

The persistence of latent HIV-1 reservoirs poses a major risk for rebound viremia, necessitating strategies that enhance the clearance of intracellular viral components. Autophagy is a critical homeostatic mechanism for clearance of damaged organelle and misfolded proteins, yet its role in HIV-1 infection is complex, as the virus often evolves mechanisms to evade autophagic degradation. In this study, we identify the lysosomal transporter SLC29A3/ENT3 as a key regulator of viral clearance. Using SLC29A3-knockdown (KD) and overexpressing (OE) HEK293-T cell models, we demonstrate that the loss of SLC29A3 significantly increases susceptibility to infection by both HIV enveloped (Clade B and C) and VSV-G enveloped HIV-1 viral particles. SLC29A3-deficient cells exhibited higher rates of host genome integration and prolonged retention of the viral capsid protein p24. Immunofluorescence microscopy revealed that the absence of SLC29A3 leads to the persistent accumulation of viral cores within endolysosomes. Conversely, SLC29A3 replenishment expedited viral clearance; a process confirmed to be autophagy-dependent through pharmacological modulation with rapamycin and chloroquine. Our findings suggest that augmenting SLC29A3 activity may provide a novel therapeutic avenue for curbing viral load by enhancing virophagy.