FIG 9.

Trehalose inhibits intracellular HIV replication in CD4⁺ T cells via induction of autophagy. (A) PHA-T cells were treated with trehalose (Tre; 100 mM, 150 mM) for 6 h. Bafilomycin cotreatment was included to confirm the increase in autophagy flux in the presence of trehalose. Cells were harvested, lysed, and analyzed for expression of LC3B lipidation by immunoblotting. (Top) Representative immunoblot showing expression of LC3B-II and ACTB. (Bottom) Relative fold change (densitometric analysis) in LC3B-II expression normalized to ACTB. (B) Uninfected and HIV-infected CD4⁺ T cells were treated with vehicle or trehalose (100 mM and 150 mM) on day 3 p.i. and incubated for 10 days. At day 10, cells were harvested and lysates were analyzed by immunoblotting with anti-LC3B, anti-RAB7, anti-LAMP1, and anti-LAMP2 and ACTB antibody. (Top) Representative immunoblots of LC3B, RAB7, LAMP1, and LAMP2. (Bottom) Densitometric analysis of uninfected CD4⁺ T cells (gray) and infected CD4⁺ T cells (upper right, red). (C) Infected T cells transfected with nonspecific control siRNA (siControl) or ATG5 siRNA were treated with 100 mM trehalose for 3 days. At 48 h post-siRNA transfection, cells were harvested and analyzed for ATG5 expression by immunoblotting (top). At 72 h post-trehalose treatment, culture supernatants were collected and analyzed for p24 release by ELISA (bottom). Data are derived from three independent donors and presented as means ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001.