Exosome-associated release, uptake, and neurotoxicity of HIV-1 Tat protein

• Published in: Journal of neurovirology 2016-12, Vol.22 (6), p.774-788
• Main Authors: Rahimian, Pejman, He, Johnny J.
• Format: Article
• Language: English
• Subjects: Astrocytes - metabolism; Astrocytes - pathology; Biomedical and Life Sciences; Biomedicine; Cell Line, Transformed; Exosomes - metabolism; Gene Expression; HEK293 Cells; HIV Long Terminal Repeat; HIV-1 - chemistry; Humans; Immunology; Infectious Diseases; Lentivirus; Lymphocytes - metabolism; Lymphocytes - pathology; Neuroglia - metabolism; Neuroglia - pathology; Neurology; Neurosciences; Primary Cell Culture; Protein Domains; Protein Transport; Retroviridae; tat Gene Products, Human Immunodeficiency Virus - chemistry; tat Gene Products, Human Immunodeficiency Virus - genetics; tat Gene Products, Human Immunodeficiency Virus - metabolism; Transcriptional Activation; Transgenes; Virology
• ISSN: 1355-0284; 1538-2443
• DOI: 10.1007/s13365-016-0451-6

HIV-1 Tat is an indispensible transactivator for HIV gene transcription and replication. It has been shown to exit cells as a free protein and enter neighboring cells or interact with surface receptors of neighboring cells to regulate gene expression and cell function. In this study, we report, for the first time, exosome-associated Tat release and uptake. Using a HIV-1 LTR-driven luciferase reporter-based cell assay and Western blotting or in combination with exosome inhibitor, OptiPrep gradient fractionation, and exosome depletion, we demonstrated significant presence of HIV-1 Tat in exosomes derived from Tat-expressing primary astrocytes, Tat-transfected U373.MG and 293T, and HIV-infected MT4. We further showed that exosome-associated Tat from Tat-expressing astrocytes was capable of causing neurite shortening and neuron death, further supporting that this new form of extracellular Tat is biologically active. Lastly, we constructed a Tat mutant deleted of its basic domain and determined the role of the basic domain in Tat trafficking into exosomes. Basic domain-deleted Tat exhibited no apparent effects on Tat trafficking into exosomes, while maintained its dominant-negative function in Tat-mediated LTR transactivation. Taken together, these results show a significant fraction of Tat is secreted and present in the form of exosomes and may contribute to the stability of extracellular Tat and broaden the spectrum of its target cells.