135 Dramatic enhancement of uptake and trafficking of HIV-1 Tat protein via modulation of endocytosis pathways

Abstract

HIV-1 Tat protein can be secreted from infected cells and taken up by other target cells. Tat uptake and trafficking has been widely investigated. This property is also exploited for drug delivery. We initially confirmed that Tat mainly entered Hela cells by clathrin-dependent endocytosis and was degraded in lysosomes, whereas chloroquine could inhibit the degradation and enhanced its trafficking to the nucleus. Interestingly, a similar effect was noted in presence of chlorpromazine (CPZ). Most likely, the uptake of Tat was shifted to caveolar endocytosis after the clathrin pathway was blocked by CPZ. However, the LTR transactivation of Tat could be enhanced by at least 50 fold when a liposome reagent was introduced into the Tat delivery system. Further evidence showed that liposome reagents could switch the Tat uptake from clathrin-dependent endocytosis to lipid raft pathway and bypass the degradation in lysosomes. This was supported by both pharmacological assays using inhibitors of caveolae-mediated endocytosis (MβCD, etc.) or microtubules (cytochalasin D & lutrunculin A) and co-localization using Tat101venus and immunstaining with anti-clathrin1, anti-caveolin1, anti-EEA1, anti-Lamp1 or anti-Rab7. Subsequently, chemical molecules carrying “+” or “−” charges were tested for blocking the Tat uptake facilitated with the liposome reagent, including macromolecules (e.g., heparin, polybrene) and small molecules (e.g., spermine). Both “+” and “-” charged molecules could inhibit the uptake of Tat, but the “+” molecules actually attached to the membrane carrying “-” charges and the “-” molecules bound to the Tat-liposome complex with “+” charges. However, the Tat-liposome complex was probably formed by hydrophobic bonds linking liposome's lipid to Tat hydrophobic domain because both Tat and the liposome reagent carried “+”charges. A peptide containing partial hydrophobic domain of Tat (CFITKGLGISYGRKK) could competitively inhibit the uptake of Tat-liposome complex. In conclusion, the liposome reagent could facilitate Tat protein to traverse the membrane by changing endocytosis pathways and significantly enhance its delivery to the nucleus in a lysosome-independent manner. These observations have important implications for elucidating the mechanism of Tat uptake and trafficking and exploring drug delivery.