Cationic liposome-mediated uptake of human immunodeficiency virus type 1 Tat protein into cells.
The human immunodeficiency virus type 1 (HIV-1) Tat protein strongly transactivates gene expression from the viral long terminal repeat (LTR) and is required for virus efficient replication. Previous studies have shown that cells scrape-loaded in the presence of purified recombinant Tat can absorb the protein in a receptor-independent fashion. Using recombinant Tat in which cysteine residues were blocked by sulfitolysis to prevent disulfide aggregation (S-Tat) we were unable to observe this phenomenon, possibly because of improper protein folding. In this study we report that the block to cellular uptake could be overcome by mixing S-Tat with a cationic liposome, Lipofectin. When mixed with Lipofectin, S-Tat effected a specific, concentration-dependent transactivation of HIV-1 LTR-directed reporter gene activity in Hela Cells. Cellular uptake was confirmed by Western blot analysis with an anti-Tat antibody. The method described utilizes cells plated in a 96-well format, requires only nanogram quantities of S-Tat protein and is much less labor-intensive than assays involving scrape-loading, making it suitable for use as a high-throughput screen for detecting Tat inhibitors. The method may have applications for the analysis of other recombinant proteins that require uptake into intact cells for determination of functionality and presents a general technique for introducing exogenous proteins into cells.
Fong
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SE
Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285-0438, USA. Fong@mail.ncifcrf.gov
Smanik
P
P
Smith
M C
MC
Jaskunas
S R
SR
D015153:Blotting, Western; D002412:Cations; D002738:Chloroquine / Q000494:pharmacology; D015696:Gene Products, tat / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
D002412:Cations; D015696:Gene Products, tat; D008081:Liposomes; D010714:Phosphatidylethanolamines; D054322:tat Gene Products, Human Immunodeficiency Virus; C020888:1,2-dielaidoylphosphatidylethanolamine; D002738:Chloroquine