LAUSR

Dear Editor, Human immunodeficiency virus type 1 (HIV-1) infection is mainly initiated on mucosal epithelial surfaces. Exposure to HIV-1 impairs the mucosal epithelial barrier, allowing viral transmission across the mucosal epithelium. Interaction of HIV-1 envelope glycoprotein gp120 with its primary receptor, cluster of differentiation 4 (CD4), and chemokine coreceptors has been implicated in the disruption of epithelial cell junctions upon HIV-1 exposure. However, the molecular details and underlying mechanisms of this disruption remain elusive. To investigate the mechanisms, we purified recombinant gp120, which was functional, as indicated by its efficient interaction with CD4 (Supplementary Data Fig. S1a–c). As T84 human colon cancer cells gradually achieved confluency, the transepithelial electrical resistance (TEER) was increased but was significantly decreased by the addition of gp120 to the culture (Supplementary Data Fig. S1d). The increase in epithelial permeability by gp120 was confirmed by the measurement of paracellular permeability (Supplementary Data Fig. S1e). By examining the localization of the tight junction marker zona occludens 1 (ZO-1) and the adherens junction marker E-cadherin, we found that gp120 impaired the integrity of tight and adherens junctions in T84, SW480, and Caco-2 human colon cancer cells (Fig. 1a, b) and HOEC human oral epithelial cells (Supplementary Data Fig. S1f, g). We then sought to investigate the mechanism by which gp120 disrupts these cell junctions. Analysis of whole-cell lysates from control and gp120-treated RKO human colon cancer cells revealed that gp120 significantly decreased the level of stathmin (Supplementary Data Fig. S2a), a microtubule-destabilizing protein. The reduction of stathmin by gp120 was confirmed by immunoblotting (Fig. 1c). This deleterious effect on stathmin was abrogated by treatment with AMD3100 (Supplementary Data Fig. S2b), a compound that antagonizes the gp120 interaction with its chemokine coreceptors. The levels of stathmin were also decreased in HCT116 human colon cancer cells, Ca9-22 human oral cancer cells, and VK2 human vaginal epithelial cells following incubation with gp120 (Supplementary Data Fig. S2c). In contrast, the levels of other microtubule-binding proteins were not significantly altered by gp120 (Supplementary Data Fig. S2d). We also found that the overexpression of stathmin could rescue both tight and adherens junctions that had been disrupted by gp120 treatment (Fig. 1d and Supplementary Data Fig. S2e). These results suggest that the perturbation of epithelial cell junctions by gp120 results from the decrease in the level of stathmin. To investigate the molecular mechanism underlying the reduction of stathmin, we examined its stability. The half-life of stathmin was decreased in the presence of gp120 (Supplementary Data Fig. S3a, b). Treatment with the autophagy-lysosome inhibitor chloroquine (CQ), but not the proteasome inhibitor MG132, ameliorated the decrease in stathmin, suggesting that stathmin is degraded by the autophagy-lysosome pathway (Fig. 1e). To test this hypothesis, we quantified fluorescent puncta corresponding to the autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3) in autophagosomes. Incubation with gp120 enhanced the formation of intracellular LC3 puncta (Fig. 1f). Accordingly, more stathmin was targeted to intracellular LC3 puncta in the presence of gp120 (Supplementary Data Fig. S3c). Immunoprecipitation analysis revealed the association of green fluorescent protein (GFP)-stathmin with Flag-p62 (Supplementary Data Fig. S3d), the receptor for cargoes destined for degradation by autophagy. Moreover, treatment with gp120 promoted the interaction between endogenous stathmin and p62 (Fig. 1g). Compared to that of the control cells, the level of stathmin was not changed by gp120 in p62-depleted cells (Fig. 1h). In addition, knocking down Atg5 or Atg6, two other autophagy regulators, prevented the gp120-induced decrease in stathmin (Supplementary Data Fig. S3e). We also found that the disruption of tight and adherens junctions by gp120 was blocked by silencing Atg5, Atg6, or p62 (Fig. 1i, j and Supplementary Data Fig. S4a–d) or by treating cells with bafilomycin A1 (BFA1), another autophagy inhibitor, or CQ (Supplementary Data Fig. S4e–h). Collectively, these data suggest that the gp120-induced autophagic degradation of stathmin contributes to the breakdown of cell junctions. We then investigated whether the gp120-induced reduction in stathmin interferes with microtubule turnover. We found that incubating Caco-2 cells with gp120 caused microtubule hyperstabilization, as evidenced by an increase in microtubule acetylation, which was abrogated by treatment with AMD3100 (Fig. 1k and Supplementary Data Fig. S5a). Similar results were obtained with oral and vaginal epithelial cells (Supplementary Data Fig. S5b, c). Microtubules in gp120-treated cells were less affected by cold shock than microtubules in the control cells, which were depolymerized, confirming the microtubule hyperstabilization induced by gp120 (Supplementary Data Fig. S5d, e). We also found that overexpression of stathmin could abrogate the microtubule hyperstabilization induced by gp120 (Fig. 1l). In addition, the gp120-induced increase in microtubule hyperstabilization was blocked by treatment with BFA1 or CQ (Supplemen-