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)