HIV-1 Vpu Downregulates Tim-3 from the Surface of Infected CD4⁺ T Cells

Along with other immune checkpoints, T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is expressed on exhausted CD4⁺ and CD8⁺ T cells and is upregulated on the surface of these cells upon infection by human immunodeficiency virus type 1 (HIV-1). Recent reports have suggested an antiviral role for Tim-3. However, the molecular determinants of HIV-1 which modulate cell surface Tim-3 levels have yet to be determined. Here, we demonstrate that HIV-1 Vpu downregulates Tim-3 from the surface of infected primary CD4⁺ T cells, thus attenuating HIV-1-induced upregulation of Tim-3. We also provide evidence that the transmembrane domain of Vpu is required for Tim-3 downregulation. Using immunofluorescence microscopy, we determined that Vpu is in close proximity to Tim-3 and alters its subcellular localization by directing it to Rab 5-positive (Rab 5⁺) vesicles and targeting it for sequestration within the trans- Golgi network (TGN). Intriguingly, Tim-3 knockdown and Tim-3 blockade increased HIV-1 replication in primary CD4⁺ T cells, thereby suggesting that Tim-3 expression might represent a natural immune mechanism limiting viral spread.IMPORTANCE HIV infection modulates the surface expression of Tim-3, but the molecular determinants remain poorly understood. Here, we show that HIV-1 Vpu downregulates Tim-3 from the surface of infected primary CD4⁺ T cells through its transmembrane domain and alters its subcellular localization. Tim-3 blockade increases HIV-1 replication, suggesting a potential negative role of this protein in viral spread that is counteracted by Vpu.