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. 2018 Mar 12:2018:7863412.
doi: 10.1155/2018/7863412. eCollection 2018.

Detection of HIV-1 and Human Proteins in Urinary Extracellular Vesicles from HIV+ Patients

Samuel I Anyanwu  1 , Akins Doherty  1 , Michael D Powell  1 , Chamberlain Obialo  2 , Ming B Huang  1 , Alexander Quarshie  3 , Claudette Mitchell  1 , Khalid Bashir  2 , Gale W Newman  1
Affiliations

Detection of HIV-1 and Human Proteins in Urinary Extracellular Vesicles from HIV+ Patients

Samuel I Anyanwu et al. Adv Virol. .

Abstract

Background: Extracellular vesicles (EVs) are membrane bound, secreted by cells, and detected in bodily fluids, including urine, and contain proteins, RNA, and DNA. Our goal was to identify HIV and human proteins (HPs) in urinary EVs from HIV+ patients and compare them to HIV- samples.

Methods: Urine samples were collected from HIV+ (n = 35) and HIV- (n = 12) individuals. EVs were isolated by ultrafiltration and characterized using transmission electron microscopy, tandem mass spectrometry (LC/MS/MS), and nanoparticle tracking analysis (NTA). Western blots confirmed the presence of HIV proteins. Gene ontology (GO) analysis was performed using FunRich and HIV Human Interaction database (HHID).

Results: EVs from urine were 30-400 nm in size. More EVs were in HIV+ patients, P < 0.05, by NTA. HIV+ samples had 14,475 HPs using LC/MS/MS, while only 111 were in HIV-. HPs in the EVs were of exosomal origin. LC/MS/MS showed all HIV+ samples contained at least one HIV protein. GO analysis showed differences in proteins between HIV+ and HIV- samples and more than 50% of the published HPs in the HHID interacted with EV HIV proteins.

Conclusion: Differences in the proteomic profile of EVs from HIV+ versus HIV- samples were found. HIV and HPs in EVs could be used to detect infection and/or diagnose HIV disease syndromes.

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Figures

Figure 1
Figure 1
Detection of HIV-1 proteins by western blot. Extracellular vesicles were isolated from four ml of urine from HIV-1+ patients and HIV-1 negative individuals by Amicon ultrafiltration (MW cutoff = 100,000 kD). The western blot is representative of 9 HIV+ and 3 HIV-negative samples (c1, c2, and c3). Recombinant HIV Nef and p24 were added as positive controls (last panels on the right). Samples were isolated in a 4–20% gradient SDS gel and transferred to a PVDF membrane. The filter was incubated with the primary antibody, pooled HIV-1 positive plasma (bottom panels), or a monoclonal anti-HIV Nef (top panels). The secondary antibody, goat anti-mouse IgG for the anti-Nef blots or rabbit anti-human IgG for the anti-HIV antibodies, conjugated to horseradish peroxidase. Super Signal West Femto was used as chemiluminescent substrate for detection.
Figure 2
Figure 2
Transmission electron microscopy of urinary extracellular vesicles. Four mls of urine was used to isolate EVs by Amicon ultrafiltration (MW cutoff = 100,000 kD). EVs were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer. Samples were stained with 1% osmium tetroxide in 0.1 M cacodylate buffer and subsequently stained with 0.5% aqueous uranyl acetate. A JEOL 1200EX transmission electron microscope (JEOL, Peabody, MA) was used for observation and photography. 1A. EVs from HIV-1 posi.
Figure 3
Figure 3
Nanosight analysis (representative analysis). (a) NTA analysis of an HIV-negative urine sample had 0.4 × 108 particles per ml (left panel) while (b) depicts an urine sample from a HIV+ patient that had 8.7 × 108 particles per ml and has a greater relative intensity profile (right panel (a) and (b)) when compared to the HIV-negative sample. The Rank Sum T test showed that HIV+ patient urine samples had more particles per ml than the negative control urine (P < 0.05).
Figure 4
Figure 4
Percentage of proteins found in HIV+ urinary EVs. FunRich analysis of the LC/MS/MS proteins from HIV+ EVs determined the most likely tissue expressing the proteins, site of expression, and the cellular component from which the protein is derived. Data is graphed as the percentage of proteins found. ∗∗ denotes significance, P < 0.01.
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References

    1. Schorey J. S., Cheng Y., Singh P. P., Smith V. L. Exosomes and other extracellular vesicles in host-pathogen interactions. EMBO Reports. 2015;16:24–43. doi: 10.15252/embr.201439363. - DOI - PMC - PubMed
    1. Schorey J. S., Harding C. V. Extracellular vesicles and infectious diseases: New complexity to an old story. The Journal of Clinical Investigation. 2016;126(4):1181–1189. doi: 10.1172/JCI81132. - DOI - PMC - PubMed
    1. Dreyer F., Baur A. Biogenesis and functions of exosomes and extracellular vesicles. Methods in Molecular Biology. 2016;1448:201–216. doi: 10.1007/978-1-4939-3753-0_15. - DOI - PubMed
    1. Février B., Raposo G. Exosomes: Endosomal-derived vesicles shipping extracellular messages. Current Opinion in Cell Biology. 2004;16(4):415–421. doi: 10.1016/j.ceb.2004.06.003. - DOI - PubMed
    1. Ciardiello C., Cavallini L., Spinelli C., et al. Focus on extracellular vesicles: New frontiers of cell-to-cell communication in cancer. International Journal of Molecular Sciences. 2016;17(2) doi: 10.3390/ijms17020175. - DOI - PMC - PubMed

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