Molecular mechanisms in the dramatic enhancement of HIV-1 Tat transduction by cationic liposomes

Abstract

Human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat) protein possesses a unique membrane-transduction property. Interestingly, Tat transduction could be dramatically increased 1000-fold based on LTR-transactivation assay when complexed with cationic liposomes (lipo-Tat), compared with Tat alone. Therefore, underlining mechanisms were explored further. Microscopy and flow cytometry showed that this effect was associated with enhanced membrane binding, large particle formation (1-2 μm) and increased intracellular uptake of Tat fluorescent proteins. Using pharmacological assays and immune colocalizations, it was found that lipid raft-dependent endocytosis and macropinocytosis were major pathways involved in lipo-Tat uptake, and actin-filaments played a major role in intracellular trafficking of lipo-Tat to the nucleus. Furthermore, we found that the Tat hydrophobic domain (aa 36-47) mediated formation of two positively charged molecules into lipo-Tat complexes via hydrophobic bonds, based on LTR-transactivation inhibition assay. Thus, the hydrophobic domain may play an important role in Tat protein uptake and be useful for intracellular delivery of biomacromolecules if coupled together with Tat basic peptide, a cell-penetrating peptide.

Figure 1.

Enhancement of Tat uptake by complexing Tat proteins with cationic liposomes. Tat uptake was monitored in cell lines stably transfected with HIV-LTR conjugated to a reporter gene, CAT or β-galactosidase (β-gal). A) LTR transactivation of Tat86 was significantly enhanced by Lipo2000 (1:500) or ChQ (60 μM) in HL3T1 cells. B) LTR transactivation of Tat101v was enhanced by Lipo2000 (1:500) or ChQ (60 μM) in TZM-bl cells. C) LTR transactivation of Tat72 was significantly enhanced by Lipo2000 (1:250) in HL3T1 cells. D) LTR transactivation was enhanced using pcDNA-Tat72-transfected supernatant (Tat72 Sup) in the presence of Lipo2000 (1:500). E) Enhancement of Tat72-LTR transactivation with Lipo2000 (1:250) was observed in SVGA-LTR-CAT cells. F) LTR transactivation of Tat86 was enhanced by different cationic liposome reagents (1:500) in HL3T1 cells. Results are representative of ≥3 experiments for each condition.

Figure 2.

Morphological characterization of fluorescent Tat proteins complexed to cationic liposomes. A) Binding of Tat101v without or with Lipo2000 (1:500) in HL3T1 cells after 20 min incubation. Lipo-Tat complexes are seen on the cell membrane (arrows). B) Binding of Tat101v without or with Lipo2000 (1:500) in TZM-bl cells after 20 min incubation. C) Membrane attachment of Tat-FITC complexed with Lipo2000 (1:200) or uncomplexed on HL3T1 and TZM-bl cells after 20 min incubation. D) Intracellular uptake of Tat-FITC with or without Lipo2000 (1:250) in HL3T1 and TZM-bl cells after incubation for 2 h and trysinization for 15 min. Colors in graphs of flow cytometry: dark gray, untreated cells; green, Tat-FITC only; red, Tat-FITC complexed with Lipo2000. View in images: ×630.

Figure 3.

Effect of endocytic blockers on Tat-LTR transactivation in the presence of Lipo2000. A) LTR transactivation was blocked partially or completely by endocytic inhibitors in HL3T1 cells treated with the lipo-Tat86 complexes (Tat86=20 ng/ml). B) LTR transactivation was blocked partially or completely by endocytic inhibitors in TZM-bl cells treated with the lipo-Tat86 complexes (Tat86=100 ng/ml). Results represent ≥3 experiments. *P < 0.05, **P < 0.01; Student's t test.

Figure 4.

Colocalization of Tat with anti-clathrin heavy chain (HC) or transferrin. A) Tat101v (green) colocalizes with anti-clathrin HC (red) in HL3T1 cells (arrows), but not in the presence of Lipo2000. B) Similar results are also found for Tat101v (green) and Alexa Fluor 568 transferrin (red) in the presence or absence of Lipo2000 in HL3T1 cells. View: ×630.

Figure 5.

Colocalization of Tat with anti-caveolin or cholera toxin B. A) Focal areas of colocalizations (arrows) are shown for Tat101v (green) and anti-caveolin (red) in both the presence and absence of Lipo2000 in TZM-bl cells. B) Tat101v (green) colocalized with Alexa Fluor 594 cholera toxin B (red) in both the presence and absence of Lipo2000 in TZM-bl cells. Magnification: ×630.

Figure 6.

Effect of trafficking blockers on the functional nuclear delivery of Tat86 in the presence of Lipo2000. A) LTR transactivation was completely or partially blocked by cytochalasin D, latrunculin A, paclitaxel and brefeldin A, but not by nacodazole and bafilomycin A1 in HL3T1 cells treated with the lipo-Tat86 complexes (20 ng/ml). B) LTR transactivation was completely or partially blocked by cytochalasin D, latrunculin A, paclitaxel, bafilomycin A1 and brefeldin A, but not by nacodazole in TZM-bl cells treated with the lipo-Tat86 complexes (100 ng/ml). Results represent ≥3 experiments. *P < 0.05, **P < 0.01; Student's t test.

Figure 7.

Inhibition of lipo-Tat uptake by macromolecules carrying positive or negative charges. A) Lipo-Tat uptake was inhibited by polybrene carrying positive charges and polysaccharides carrying positive charges (DEAE-dextran) or negative charges (heparin and dextran sulfate; Tat86=20 ng/ml). B) Inhibition of negative macromolecules on Tat uptake was abolished by pretreating the cells for 30 min, followed by a washing step, and then adding lipo-Tat86 complexes (50 ng/ml); however, only slight or no influence was found on the inhibition using the same protocol for positive macromolecules or following preincubation of negative/positive macromolecules with lipo-Tat complexes. C) Uptake of the lipo-Tat complexes was inhibited by poly-l-lysine hydrobromide (P-l-lysine) with different molecular weights (carrying positive charges; Tat72=400 ng/ml). Results represent 3 experiments.

Figure 8.

Role of Tat hydrophobic domain on the uptake of the lipo-Tat complexes. A) Diagram of HIV-1 Tat protein and sequence of its hydrophobic domain. B) Tat86 uptake and its LTR-transactivation was inhibited by preincubating Lipo2000 with Tat hydrophobic peptide for 30 min before complexing Tat86 (200 ng/ml), but no significant inhibition was found if it was replaced by an SC hydrophobic peptide. Results represent 3 experiments. C) No significant inhibition of HIV-1 JRCSF replication was observed when TZM-bl cells were pretreated with Tat or SC hydrophobic peptides complexed to Lipo2000 or uncomplexed.