Tetraspanin blockage reduces exosome-mediated HIV-1 entry

Archives of Virology, Feb 2018

HIV-1 is one of the most studied retroviruses. The role of exosomes in HIV-1 entry and pathogenesis are beginning to be appreciated. Exosomes can incorporate host proteins that are also contained in viruses (e.g., tetraspanins).

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Tetraspanin blockage reduces exosome-mediated HIV-1 entry

Archives of Virology Tetraspanin blockage reduces exosome‑mediated HIV‑1 entry Brian Sims 0 1 2 4 Anitra L. Farrow 0 1 2 4 Sparkle D. Williams 0 1 2 4 Anju Bansal 0 1 2 4 Alexandre Krendelchtchikov 0 1 2 4 Qiana L. Matthews 0 1 2 4 0 Division of Infectious Diseases, Department of Medicine, University of Alabama at Birmingham , Birmingham , USA 1 Center for AIDS Research, University of Alabama at Birmingham , Birmingham , USA 2 Division of Neonatology, Departments of Pediatrics, Neurobiology and Cell, Developmental and Integrative Biology, University of Alabama at Birmingham , Birmingham , USA 3 Qiana L. Matthews 4 Microbiology Program, Department of Biological Sciences, College of Science, Technology, Engineering and Mathematics, Alabama State University , Montgomery, AL 36104 , USA HIV-1 is one of the most studied retroviruses. The role of exosomes in HIV-1 entry and pathogenesis are beginning to be appreciated. Exosomes can incorporate host proteins that are also contained in viruses (e.g., tetraspanins). Abbreviations HEK 293 Human embryonic kidney cells TIM4 T cell immunoglobulin and mucin protein 4 NTA Nanoparticle tracking analysis HIV-1 Human Immunodeficiency virus-1 CD cluster of differentiation IMC infectious molecular clone LucR Renilla luciferase HAND HIV-associated neurocognitive disorder Handling Editor: Li Wu. Introduction Human immunodeficiency virus-1 (HIV-1) is one of the most studied retroviruses; however, there is still a wealth of information to be learned about this virus’s entry, lifecycle and pathogenesis. The role of exosomes in HIV-1 entry and pathogenesis is beginning to be appreciated. Exosomes Brian Sims and Anitra L. Farrow contributed equally to this work. are small extracellular vehicles, which traffic nucleic acids (RNA, microRNA, and DNA), proteins, and lipids. Exosomes are secreted by most cell types into the extracellular milieu and then internalized into the recipient cells. It has been well documented that exosomal composition reflects the donor cells in which they are generated. Exosomes can incorporate host proteins that are also contained in viruses (e.g., tetraspanins) [ 1, 2 ]. Furthermore, exosomes appear to affect viral infection [ 3–9 ]. It has been previously demonstrated that blocking cluster of differentiation (CD) 9 and CD81 tetraspanins, which are commonly expressed on exosomes, resulted in significant decreases in the exosomal uptake efficiency in dendritic cells [10]. Regarding HIV-1, the role of exosomes in pathogenesis is complex. Studies have shown that exosomes may promote or inhibit HIV-1 infection [ 11, 12 ]. The overlap between HIV-1 and exosome biogenesis within an infected cell suggests that HIV-1 products, including RNA and proteins may be encased within exosomes or contaminate exosome preparations from HIV-1 infected fluids [ 13–15 ]. We are just beginning to unravel the complex nature of exosomes and HIV-1 interactions via lipids, phospholipids and proteins [ 16, 17 ]. Our group has recently shown that HIV-1 entry into human immune cells is enhanced by exosome-mediated trafficking [9]. This effect was illustrated with exosomes derived from human plasma, human breast milk, mouse neural stem cells and human lung carcinoma cells. Furthermore, our study demonstrated that HIV-1 and exosome interactions were mediated partially through binding of the T cell immunoglobulin and mucin protein 4 (TIM4) to the viral envelope [ 9 ]. In this study, we demonstrated that exosomes can enhance HIV-1 entry into human T and monocytic cell lines via exosomal tetraspanin proteins CD81 and CD9. Materials and Methods Cell culture Human CD4+ lymphoblastoid T cell line (line A3R5.7) was a gift from the UAB CFAR Virology core. These cells were commercially received from the NIH AIDS Research and Reference Reagent Program and subsequently genetically modified. A3R5.7 cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated, exosome-free fetal bovine serum, 2 mM l-glutamine, penicillin (100 U/mL), streptomycin (100 μg/mL) (Thermo Fisher Scientific, Waltham, MA, USA), and 1 mg/mL geneticin (G418; Thermo Fisher Scientific). Human monocytic cells (line THP2574) were maintained in similar medium but without geneticin [ 18, 19 ]. All other cell lines were purchased from American Type Culture Collection. Exosome purification Isolation of human embryonic kidney cells (HEK 293)‑derived exosomes Cell line HEK293 was grown in DMEM-F12 complete medium containing exosome-free fetal bovine serum to ~80% confluency. In brief, cells were centrifuged at 5,000 rpm for 10 min at 4°C using a Sorvall RT600 centrifuge with a swinging bucket rotor (Thermo Fisher Scientific). The supernatant was clarified by filtration through a 0.22 μm filter and centrifuged at 13,200 rpm for 70 min at 4°C using an SW41T1 swinging rotor in a Beckman Coulter (Brea, CA, USA) Optima L-70K ultracentrifuge for exosome collection [ 8, 9, 20 ]. Exosomes were resuspend (...truncated)


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Brian Sims, Anitra L. Farrow, Sparkle D. Williams, Anju Bansal, Alexandre Krendelchtchikov, Qiana L. Matthews. Tetraspanin blockage reduces exosome-mediated HIV-1 entry, Archives of Virology, 2018, pp. 1683-1689, Volume 163, Issue 6, DOI: 10.1007/s00705-018-3737-6