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

1. Introduction

Extracellular vesicles (EVs) are membrane bound vesicles, between 30 nm and 1 [micro]m in size, are secreted into blood, urine, saliva, semen, and other bodily fluids, and have been suggested as a potential source of biomarkers for disease progression [1, 2]. These EVs, microparticles and/or exosomes, are secreted by cells normally or while they are undergoing stress or apoptosis [3] and contain proteins, mRNA, and miRNA [4] that are involved in cell to cell communication, transfer of antigens to cells, and intracellular communication. EVs are described in cancer disease pathogenesis [5] in HIV infection [6], other viral infections [7], and other disease states such as cardiovascular, renal, liver, and metabolic disease [8-11].

EVs from urine are an attractive noninvasive source for biomarkers of diseases [12,13]. In healthy individuals, protein only accounts for 0.01% of urine components; however, in certain disease states, the protein content and EV numbers can increase in urine [12-16]. The glomerular capsule filters blood that is passed into the renal tubule and accounts for thirty percent of the urinary protein content [14-16]. The remaining seventy percent of proteins in urine is derived from the kidney [17], and thus, urinary EVs are comprised of both renal and efferent components.

HIV proteins are detected in EVs of HIV+ patients and HIV Nef is the most prevalent protein found [18-21]. Other reports of HIV proteins in EVs are from in vitro transfected or HIV infected cultured cells and are not from HIV+ patient samples [6,18,19, 22, 23].

Biomarkers in urinary EVs are suggested for use in the diagnosis of many disease states [12,13,24-30]. The objectives of this study were to determine the differences in proteins from urinary EVs from HIV+ patients and HIV- individuals using proteomics and mass spectrometry. The analysis of more patient samples could identify specific EV urinary proteins as biomarkers of HIV infection, treatment efficacy, and/or disease progression.

2. Methods

2.1. Sample Collection. Urine was collected from thirty-five (35) HIV+ patients and twelve (12) HIV- individuals in sterile collection cups. The subjects were recruited from clinics in the metropolitan Atlanta area, GA. Patient demographics are described in Table 1. The study was approved by the Institutional Review Board of Morehouse School of Medicine and written informed consent was obtained from all participants.

2.2. EV Isolation. Urine samples were centrifuged at 1000 xg to remove cells and sediment then frozen at -80[degrees] C. Samples, 4 ml, were thawed and the EVs isolated followed by centrifugal filtration using Amicon Ultra-4 100 kDa centrifugal filter unit (Millipore, Billerica, MA), at 3000 xg for 15 minutes at 4[degrees]C. The retentate, containing EVs, was collected from the top of the filter and resuspended in 200 [micro]l phosphate buffered saline (PBS) for use in the transmission electron microscopy and tandem mass spectrometry (LC/MS/MS) analysis.

2.3. Transmission Electron Microscopy Analysis. Transmission electron microscopy (TEM) was used to identify EVs in two HIV-1 positive and two HIV-1 negative samples. Urinary EVs were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 hours at 4[degrees]C followed by 2 washes with 0.1 M cacodylate buffer, 5 minutes each. Samples were stained with 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 hour at 4[degrees]C followed by 2 washes with the cacodylate buffer and 3 washes with deionized water, 5 minutes each. Samples were subsequently stained with 0.5% aqueous uranyl acetate for 2 hours at room temperature and subsequently viewed with a JEOL 1200EX transmission electron microscope (JEOL, Peabody, MA).

2.4. Nanoparticle Tracking Analysis (NTA). Urine samples from HIV-negative (n = 8) and positive individuals (n = 11), 15 ml, were centrifuged at 300 xg for 10 min at 4[degrees]C to remove cell debris. The supernatant was collected and centrifuged at 16,500 xg for 20 min at 4[degrees]C and the supernatant collected and ultracentrifuged at 120,000 xg at 4[degrees]C for 1.5 hr. The pellet was resuspended in 500 [micro]l of PBS. The size and quantification of the EVs were analyzed using the NanoSight NS500 (NanoSight NTA 2.3 Nanoparticle Tracking and Analysis Release version build 0025). Particles were automatically tracked and sized based on Brownian motion and the diffusion coefficient. The NTA measurement conditions were temperature 21.0 +/- 0.5[degrees]C, viscosity 0.99 +/- 0.01 cP, frames per second 24.99-25, and measurement time 30 s. The detection threshold was similar in all samples. Two recordings were performed for each sample.

2.5. Mass Spectrometry Analysis. Thirty-five (35) HIV+ and twelve (12) HIV- EV samples were lysed and trypsinized and the sequence of peptides was determined by tandem mass spectrometry (LC/MS/MS), using an LTQ Ion Trap Mass Spectrometer (Thermo Fischer Scientific, Waltham, MA). Peptides were first reduced in DTT 10 mM at 56[degrees]C for at least 30 min and alkylated with 15 mM iodoacetic acid for 30 min at room temperature in the dark. Samples were then digested with mass spectrometry grade trypsin 20 ng/[micro]l for 4 hours at 37[degrees]C. Just before analysis, the sample was acidified by the addition of formic acid to 0.1%. Peptides were separated by reverse phase HPLC (Agilent) on a 0.5 x 75 mm C-18 column (Michrom) at a flow rate of 500 nl/min using a linear gradient of acetonitrile (5-35%) over 100 min. Ions were directly introduced by nanospray and spectra were collected using Xcalibur 2.0 software using an intensity threshold of 200 counts. The resulting spectra were analyzed using Bioworks 1.1 software to search a hybrid Human-HIV database created from the complete nonredundant peptide database from NCBI. The threshold for inclusion in the search is a minimal S/N ratio of 3. False discovery rates were determined and set based on the control HIV- samples. An initial protein identification list was generated from matches with an Xcorr score versus charge state of 1.0 (+1) 1.5 (+2) and 1.7 (+3) and consensus scores greater than 10.0.

Bioinformatics techniques for analysis of HIV EV proteins were used on the LC/MS/MS detected proteins [31]. Functional enrichment analysis was performed using FunRich (Functional Enrichment analysis tool, http://funrich .org/index.html) [32] against a human database to detect proteins involved in biological processes, cellular components, sites of expression, and biological pathways. Only processes with a P value < 0.05, using the Benjamini-Hochberg False Discovery rate, were reported. The human proteins detected were compared to the top 100 EV proteins in ExoCarta (http://exocarta.org/exosome_markers_new) [33, 34], sixty EV proteins in the EV array [35], and proteins identified in EVs from HIV infected lymphocytic cells [36].

Pathway analysis comparing HIV+ samples with CD4+ T cells greater than 300 (n = 15) to those with less than 500 (n = 15) and HIV high VL, greater than 200 copies (n = 10), compared to HIV low viral loads, less than 200 copies (n = 10), was done using Pathway Studio version 11.4 Mammal Plus (Elsevier, Inc., Atlanta, GA). Gene Set Enrichment Analysis (GSEA) was used to identify the top 10 curated pathways for the proteins in the each of the patient groups. No comparisons were done between patients not on ART or undergoing ART because there was only one patient not on ART.

The HIV proteins, Nef, Vpr, Vpu, and Vif, were searched using the HIV-1 Human Interaction database (https://www .ncbi.nlm.nih.gov/genome/viruses/retroviruses/hiv-1/interactions/). This database contains all the known, published interactions of HIV-1 gene products with human proteins [37]. Proteins from the search were compared to the human proteins detected in the HIV EVs.

2.6. Western Blot Analysis. To validate the presence of HIV proteins in urinary EVs, western blot analysis (WB) was performed on twenty (20) randomly selected HIV+ and three (3) HIV- control urine samples. Recombinant HIV-1 Nef and HIV-1 p24 were used as positive controls, while HIV-negative urine and HIV-positive filtrate were used as negative controls. Samples were heated at 85[degrees] C for two minutes in a tris-glycine SDS sample buffer, were loaded into a 4-20% TGX gradient gel (Bio-Rad, Hercules, CA), and run for 50 mins at 200 V. A semidry transfer unit (Hoefer Scientific, Holliston, MA) was used to transfer the separated proteins onto a PVDF membrane (Bio-Rad) at 15 V for 50 mins. The filter was blocked for nonspecific binding using 5% nonfat dry milk in 1x tris buffered saline (TBS) with Tween 20. The membrane was incubated overnight in pooled plasma from twenty HIV+ patients as the primary antibody at a 1 : 500 dilution and rabbit anti-human IgG conjugated HRP antibody (1: 1000, Bio-Rad, Hercules, CA) was used as secondary antibody. Super Signal West Femto (Thermo Fischer Scientific, Waltham, MA) was used as a chemiluminescent substrate for detection. The membrane was developed and imaged using the LAS 4000 biomolecular imager (GE Healthcare Life Sciences, Pittsburgh, PA). Recombinant HIV-1 Nef and p24 WB analyses were detected using anti-Nef and p24 monoclonal antibodies (1: 500, EMD Millipore, Billerica, MA) and anti-mouse IgG conjugated HRP antibodies (1: 1000, Bio-Rad, Hercules, CA) were used.

2.7. HIVp24 ELISA. Twenty-six (26) HIV+ and eleven (11) HIV- urine samples were tested for the presence of HIV p24 by ELISA (ImmunoDX, Woburn, MA).

3. Results

3.1. HIV Proteins Are Present in Urinary EVs of HIV-Positive Patients. LC/MS/MS mass spectrometry HIV EV protein results are presented in Table 2. Urinary EV proteins meeting the false discovery rate and Xcorr score criteria as HIV-1 proteins included Nef, Gag, Pol, Protease, gp120, gp160, gp41, Rev, reverse transcriptase, Tat, Vif, Vpr, and Vpu. All HIV+ urine samples (n= 35) contained at least one HIV-1 protein in EVs, while no HIV proteins were found in the HIV- samples (n = 12) (Table 3). HIV-1 Nef was detected in twenty-six of thirty-five (26 of 35) (74.3%) HIV+ urine samples. Three (3) patients' urine samples, #173, #174, and #196, were tested 203, 311, and 35 days, respectively, after their first EV sample was analyzed. No difference in the HIV proteins detected in sample #196, 35 days after his previous sample, was found. #173's sample, tested 203 days after the first analysis, had a similar profile, except that Rev and Tat were not detected. In addition, #174's EVs examined 311 days after the first sampling found Rev and RT missing from the profile.

HIV p24 antigen was only detected in five of thirty-five (5 of the 35 patient) (14%) samples by LC/MS/MS, but of the twenty-six (26) HIV+ and eleven (11) HIV-negative samples tested by ELISA, no p24 was detected. There was no statistical correlation of the number of HIV proteins detected with CD4+ T cell counts, viral loads, or ART therapy.

Validation by WB analysis using polyclonal pooled patient serum and monoclonal antibodies against HIV Nef and HIV p24 indicated the presence of HIV proteins. Figure 1 is a WB using polyclonal pooled HIV+ serum used as the detection antibody. All the HIV+ patient samples contained HIV-1 proteins and the top panel shows patient samples reacting to anti-HIV Nef. HIV+ urine samples, 7 of 9 (77.7%), showed HIV-1 Nef bands at 27 kD.

3.2. TEM and NTA Analysis of EVs. TEM analysis of urine from HIV+ patients showed multiple EVs, ranging in size from 50 nm to 300 nm (Figure 2(a)), while two HIV-negative controls had fewer EVs present (Figure 2(b)). NTA analysis showed that there were significantly more EVs from HIV+ patients than healthy controls, 4.96 [+ or -] 0.0733 and 3.69 [+ or -] 0.075, respectively (P < 0.05). No significant differences were found in the size of the EVs, 110-227 nm for HIV-negative donors and 54-448 nm HIV+ samples. Representative Nanosight analyses for HIV-negative and HIV+ urine samples are shown in Figure 3.

3.3. Human Proteins in HIV+ and Negative EV Urine Samples. EV proteins from the HIV+ patients, 14,475, which entered into FunRich, functional enrichment analysis software, showed 29.44% or 1,932 proteins were associated with exosomes (Table 4). These EV identified proteins were compared to top 100 EV proteins in the ExoCarta database with 83% matching (http://exocarta.org/exosome_markers_new) [33], 22 EV proteins in the EV array [35] were similar, and 7 of 14 EV proteins identified in exosomes from HIV infected lymphocytes [36] were found and are highlighted in Table 4. Exosomal proteins found in the control samples are listed in Table 5.

The GO results of the FunRich analysis of the EVs from the HIV+ samples are summarized in Table 6 and Figure 4. The top five (P < 0.01) EV sites of expression were endothelial cells, plasma, liver, serum, and kidney and the most significant cellular components were lysosomes, exosomes, membranes, plasma membranes, the nucleus, and the cytoplasm (P < 0.01) (Figure 4). The top five ontologies (Table 6) were protein serine/threonine kinase activity, catalytic activity, GTPase activator activity, guanyl-nucleoside exchange factor activity, and cell adhesion molecule activity (P < 0.0001), the top biological process was regulation of nucleobase, nucleoside, and nucleic acid (P < 0,0001), and the most prominent biological pathway was integrin cell surface interactions (P < 0.03).

LC/MS/MS identified 15,571 proteins in EVs from HIV+ patients with CD4+ T cells greater than 300,2,115 from CD4+ T cells less than 300,15,028 proteins from patients with low VL, and 2486 from patients with high VLs. Pathway analysis was similar between EV proteins from patients with greater than 300 CD4+ T cells and low VLs and different between the low CD4+ T cells and high VLs (summarized in Table 7). The pathways found are detailed in Supplementary Material 1. Interleukin proteins detected were IL10, IL10RA, IL16, IL17RC, IL18, IL18BP, IL1RAP, IL1RL2, IL1RN, IL33, IL4I1, IL6, and IL6ST. Immunomodulatory molecules, HOXB4, CD81, CD9, TGF-[beta]1, IDO, Notch1, ADAM17, Rab4, and HGF, were also found by LC/MS/MS in addition to MHC Class I and II antigens.

The HIV-1 Human Interaction database search found that HIV Nef interacted with 559 EV proteins of 770 total human proteins (72.6%); HIV Vpr interacted with 437 EV of 598 human (73.1%); HIV Vif interacted with 162 EV of 310 human (52.2%); and HIV Vpu interacted with 165 EV of 244 human proteins which were found in the HIV+ EVs (67.6%) (see Supplementary Material 2, including PMIDs for references).

Functional analysis of the control EVs are listed in Table 8. The major sites of expression were cervicovaginal fluid, neutrophils, and gastric juice (P < 0.0001). The most significant ontologies were molecular function of the proteins and defense/immunity protein activity and principal biological processes were immune response, signal transduction, cell communication, and antigen presentation (P < 0.0073).

Only sixty-four (64) proteins overlapped between the HIV+ and control EV samples and are listed in Table 8. The top fourteen (14) GO ontologies for cellular components include extracellular exosome, extracellular region, extracellular space, hemoglobin complex, and blood microparticle (P < 0.001, Table 9), GO ontologies for molecular function were heparin binding, ion gated activity, and oxygen transporter activity, and the most significant biological processes found were response to yeast, defense response to fungus, macrophage chemotaxis, negative regulation of growth of symbiont in host, oxygen transport, and hydrogen peroxide catabolic process.

4. Discussion

This is the first report of the detection of urinary EVs containing HIV and human proteins from HIV+ patients by mass spectrometry and western blot. EVs provide intercellular communication to cells through the delivery of their cargo, nucleic acids, miRNAs, and proteins, to recipient cells reviewed in [3]. Previous studies have found EVs in plasma of HIV+ patients but did not describe HIV or human proteins within them. Others have described EVs containing HIV proteins but these results were from in vitro HIV infected cell cultures and not from HIV+ patients [18, 20, 22, 23, 36, 38-47]. This study details both the HIV and human proteins found in urinary EVs from HIV+ patients.

According to the International Society for Extracellular Vesicles (ISEV), the minimal requirements for EVs or their presence in samples includes the simultaneous detection of transmembrane proteins and cytosolic proteins with membrane/receptor binding abilities, while major cell organelles are absent [48]. LC/MS/MS analysis identified these proteins and functional enrichment analysis determined a significant number which were of exosomal origin in both the EVs in HIV+, 1,932, and HIV-, only 37 TEM analysis of HIV+ and HIV- urine showed pleiotropic membrane bound vesicles in both groups' urine samples and NTA analysis showed particles ranging in size from 50 nm to 300 nm in both groups, although the HIV+ samples had significantly more particles than uninfected samples. Other studies have found increased numbers of EVs in the plasma of HIV+ patients [43, 49]. Proteins from both the HIV+ and HIV- individuals were significantly associated with exosomal proteins, further substantiating our hypothesis that urine from HIV+ patients contains EVs (Table 10). The FunRich analysis of the sites of expression showed that a significant number of proteins were associated with the endothelium, plasma, serum, kidney, liver, and lung. These findings suggest that EVs from HIV+ patients may be filtered from these sites and concentrated in urine.

HIV has previously been detected in the urine of HIV+ patients; however, it was shown that HIV virions are associated with cell pellets and not in centrifuged urine [50, 51]. p24 is found in replicative HIV infectious virions but was not found in twenty-six of our HIV+ samples by ELISA and only five of thirty-five HIV+ EV urine samples had detectable p24 by LC/MS/MS analysis. p24 in urine pellets is derived from mononuclear cells but was found in only 3 of 80 analyzed samples [51]. This represents a low sensitivity, primarily because the HIV-1 p24 protein is not always present during advanced stages of HIV infection. To further confirm that these HIV proteins were from EVs, we tested the filtrate from ultracentrifugation (MW cutoff 100,000 kD) of HIV-positive urine, and no HIV proteins were present. We did not, however, perform an HIV infectivity assay, MAGI, on the isolated urinary EVs, and thus cannot be totally confident that HIV virions were not present in the EVs. HIV proteins in urinary EVs may be the result of a nonproductive HIV infection in the kidney [52-56] and/or EVs filtered from blood [21,49, 57]. The type of HIV protein in the EVs remained relatively constant as demonstrated by the resampling of two patients, 203 and 311 days, after the first sample that had similar results. The identification of HIV proteins in urinary EVs may be a potential noninvasive diagnostic tool to monitor HIV disease states as well as treatment efficacy.

Different proteins and pathways were found in EVs from (1) CD4+ T cell > 300 versus <300 and (2) VLs < 200 versus >200 copies. It is interesting that EVs from HIV+ patients with low VLs and high CD4+ T cells, usually indicative of better health, had more proteins detected than EVs from high VLs and low CD4+ T cells (high VLs = 2486 vrs low = 15028; low CD4+ T cells = 2115 versus high CD4+ = 15761). These groups also had overlapping pathway results; however, proteins from high VLs and low CD4+ T cells did not have similar pathway results. Further comparison and analysis of the EV protein profile between the low VL/high CD4+ T cells and high VL/low CD4+ T cells may reveal more mechanisms involved in the evolving pathology of HIV infection.

Proteins contained in EVs can both enhance and inhibit host responses from innate, inflammatory, and adaptive reactions. Proteins from HIV+ patients showed a predominantly immunosuppressive profile. IL10 is a Th2 cytokine that downregulates macrophage function and inhibits T cell proliferation while IL6 can stimulate IL10 production and inhibit the effects of TNF-a and IL1. Both these cytokines were present in the EVs from HIV+ patients while TNF-[alpha] and IL1 were not detected suggesting an immunomodulatory effect may be elicited by the EVs. Other immune downregulating factors, IDO, hOxB4, HGF, and TGF[beta]1, were found. IDO [58], HLA-G [59], and HGF [60] can inhibit natural killer cell activation which was one of the top biological processes found in the pathway analysis of the EV proteins in patients with high cD4+ T cells and low VLs. TGF[beta]-1, an inhibitor of immune function, is induced by HIV Tat [61] and is a mediator of immune suppression in HIV infection [62-64]. These proteins were found in EVs from HIV+ patients while proinflammatory cytokines were not. New studies show that HIV+ nonprogressors have lower plasma TGF[beta]-1 and IL10 than patients with progressive disease [65] and it is possible that EVs may sequester TGF[beta]-1 and IL10 and remove them from circulation. The presence of over 16 different MHC Class I and II antigens in the EVs from HIV+ patients may support the hypothesis that this mechanism is used by intracellular pathogens to evade the immune response by decreasing cytotoxic T cell activity [66]. Herpes Simplex Virus-1 binds to HLA-DR inhibiting antigen presentation that leads to immune evasion [67]. Future studies should focus on the correlation of the concentration of these factors to HIV+ patients' clinical status.

In this study, we showed that structural, regulatory, and accessory HIV proteins could be detected in urinary EVs of HIV+ patients. Our WB analysis using polyclonal and monoclonal antibodies confirmed the presence of HIV proteins in the EVs from HIV+ patients. The most prevalent protein was HIV Nef. EVs from both in vitro and patient samples have been previously reviewed in [6]. HIV Nef induces an alternative pathway for TNF induction utilizing Notch-1, ADAM17, and Rab4+, all found in EVs from HIV+ patients, which leads to high plasma TNF levels [68]. Whether the isolation of these factors in EVs represents a diminishing or enhancement of TNF production remains to be examined.

The HIV Human Interaction database found significant interactions between HIV Nef, Vpr, Vif, and Vpu and human proteins. Serine/threonine protein kinases are important in T cell receptor signaling [69]. These kinases as well as CD4 and MHC antigens were found in EVs from the HIV+ samples; however, further studies are needed to determine the mechanisms involved with EV function in HIV infections. Cell adhesion molecules, ICAM, VCAM, and PECAM, were also found in the EVs from patients. Others have reported these molecules are present in HIV+ blood samples and may represent biomarkers from inflamed endothelium due to HIV infection [70].

One of the limitations of this study was a small sample size of specific HIV syndromes such as comorbidities, AIDS, HIV-associated nephropathy, and HIV-associated dementia as well as patients on or naive to antiretroviral therapy. Increasing the numbers of HIV+ patients in these categories may allow us to determine whether specific HIV proteins as well as human proteins in urinary EVs could be associated with these conditions. Future studies will also quantify the amount of HIV proteins as well as human proteins to determine if a correlation exists between different HIV conditions and the amount of proteins detected.

HIV infection is usually detected by antibodies to HIV and can take up to three months to develop or by measuring VLs in blood whereas we can detect HIV-1 proteins in urinary EVs. In summary, urinary proteins in EVs from HIV+ patients may allow a noninvasive method to (1) rapidly screen for infection and identification of patients eligible for antiretroviral treatment (ART); (2) monitor ART treatment efficacy; and (3) diagnose HIV comorbidities.

https://doi.org/10.1155/2018/7863412

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

The authors acknowledge Jane Chu and Mahfuz Khan of Morehouse School of Medicine for technical assistance and Dr. Douglas Paulsen for his support and editorial suggestions. This study was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award no. UL1TR000454. Other funds were received from the Minority Biomedical Research Support (MBRS) of the Research Initiative for Scientific Advancement (RISE) Program 5R25GM058268 funded by NIGMS and NIH Research Endowment S21MD000101 funded by the National Institute on Minority Health and Health Disparities (NIH/NIMHD). The authors also acknowledge the Research Centers in Minority Institutions (RCMI) G12 funded by the NIH/NIMHD, #8G12MD0076202. MEB Core facility was constructed with support from the Research Facilities Improvement Grant C06 RR18386 from NIH/NCRR. The newly renovated Core Resources space was funded by G20 RR031196 from NIH/NCRR. The R-CENTER was funded by Grant no. U54MD007588 from NIH/NIMHD and NIH/NCRR 5P20R R0111044 pilot for study support.

Supplementary Materials

Supplementary 1. Top 10 biological function pathways using Pathway Studio 11 Mammal Plus, Elsevier, Inc., for HIV+ EV proteins from HIV+ patients with (1) CD4+ T cells greater than 300, (2) CD4+ T cells less than 300, (3) viral loads less than 200 copies, and (4) viral loads greater than 200 copies.

Supplementary 2. EV HIV protein interactions (Nef, Vif, Vpr, and Vpu) with human proteins identified using HIV-1 Human Interaction database (https://www.ncbi.nlm.nih.gov/ genome/viruses/retroviruses/hiv-1/interactions/). The file includes the gene symbol, human protein name, interaction keywords, protein accession ID, and PMID of references citing the interaction.

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

(1) Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, GA, USA

(2) Department of Medicine, Morehouse School of Medicine, Atlanta, GA, USA

(3) Clinical Research Center, Morehouse School of Medicine, Atlanta, GA, USA

Correspondence should be addressed to Gale W. Newman; gnewman@msm.edu

Received 25 October 2017; Revised 7 January 2018; Accepted 4 February 2018; Published 12 March 2018

Academic Editor: Jay C. Brown

Caption: 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 HIVnegative 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.

Caption: 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.

Caption: Figure 3: Nanosight analysis (representative analysis). (a) NTA analysis of an HIV-negative urine sample had 0.4 x [10.sup.8] particles per ml (left panel) while (b) depicts an urine sample from a HIV+ patient that had 8.7 x [10.sup.8] 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).
Table 1: Patient demographics.

Characteristics                  HIV-positive (N = 35)

Age (median [+ or -] IQR)         41.5 [+ or -] 14.25
Sex (n, %)
  Male                                 25 (71.4%)
  Female                               10 (28.6%)
Race (n, %)
  African American/Black                28 (80%)
  White                                 7 (20%)
  Hispanic                                 --
  Asian                                    --
Viral loads (copies/ml)              50 [+ or -] 0
  (median [+ or -] IQR)
CD4+ T cell (cells/[micro]l)       66.5 [+ or -] 46.5
  (median [+ or -] IQR)
Antiretroviral therapy (n, %)          34 (971%)

Characteristics                  HIV-negative (N = 12)

Age (median [+ or -] IQR)            59 [+ or -] 18
Sex (n, %)
  Male                                 7 (58.3%)
  Female                               5 (41.7%)
Race (n, %)
  African American/Black               12 (100%)
  White                                    --
  Hispanic                                 --
  Asian                                    --
Viral loads (copies/ml)                    --
  (median [+ or -] IQR)
CD4+ T cell (cells/[micro]l)               --
  (median [+ or -] IQR)
Antiretroviral therapy (n, %)              --

Table 2: LC/MS/MS analysis of EV HIV proteins.

Accession     # AAs   MW [kDa]   Calc, pi

gi3 8491705    192      22.7       10.1
gi73913089     104      11.7       10.1
gi58374258     869      98.1       8.8
gil83197180    404      45.8       8.4
gi255984636    160      18.1       5.3
gi256012108    114      13.5       5.7
gi9756252      524      60.4       8.7
gi67082579     191      22.3       9.4
gi2290009      852      96.7       8.5
gil67886806    25       2.7        8.7
gi23344577     99       10.6       9.4
gi4324808     1437     161.9       8.3
gi222533599    73       8.0        9.1
gi71060450     206      23.7       6.3
gi37935985     85       10.3       4.8
gil08860432    870      98.8       8.5
gill4801226    209      24.4       10.1
gi22596451     341      38.6       8.0
gil83200570    342      38.7       9.2
gi34786230     176      19.8       9.6
gil002239      104      11.5       8.9
gi77168129     95       11.3       7.6
gi222532593    129      14.7       8.4
gi219688191    132      14.9       10.2
gi255687141    288      32.1       7.7
gi222532161    132      15.0       9.5
gi37934078     573      65.0       9.0
gi405003       207      23.1       7.7
gi54792352     213      23.7       5.6
gi3885826      132      14.9       9.6

Accession     Description

gi3 8491705   Vif protein [human immunodeficiency virus 1]
gi73913089    Gag protein [human immunodeficiency virus 1]
gi58374258    Envelope glycoprotein [human immunodeficiency virus 1]
gil83197180   Pol protein [human immunodeficiency virus 1]
gi255984636   Reverse transcriptase [human immunodeficiency virus 1]
gi256012108   Nef protein [human immunodeficiency virus 1]
gi9756252     Pol precursor [human immunodeficiency virus 1]
gi67082579    Reverse transcriptase [human immunodeficiency virus 1]
gi2290009     Envelope glycoprotein [human immunodeficiency virus 1]
gil67886806   Rev protein [human immunodeficiency virus 1]
gi23344577    Protease [human immunodeficiency virus 1]
gi4324808     Gag-pol polyprotein [human immunodeficiency virus 1]
gi222533599   Env C2V3 protein [human immunodeficiency virus 1]
gi71060450    Negative factor [human immunodeficiency virus 1]
gi37935985    Vpu protein [human immunodeficiency virus 1]
gil08860432   gpl60 [human immunodeficiency virus 1]
gill4801226   Tat protein [human immunodeficiency virus 1]
gi22596451    Truncated envelope glycoprotein [human
              immunodeficiency virus 1]
gil83200570   Truncated poi protein [human immunodeficiency virus 1]
gi34786230    gpl20 protein [human immunodeficiency virus 1]
gil002239     Envelope glycoprotein, v3 region [human
              immunodeficiency virus 1]
gi77168129    Vpr protein [human immunodeficiency virus 1]
gi222532593   Gag pl7 protein [human immunodeficiency virus 1]
gi219688191   Matrix protein [human immunodeficiency virus 1]
gi255687141   Integrase [human immunodeficiency virus 1]
gi222532161   Gag pl7 protein [human immunodeficiency virus 1]
gi37934078    Gag-pol fusion polyprotein [human immunodeficiency
              virus 1]
gi405003      gpl20 [human immunodeficiency virus 1]
gi54792352    Gag polyprotein [human immunodeficiency virus 1]
gi3885826     pl7 matrix [human immunodeficiency virus 1]

Accession     [SIGMA]    [SIGMA]#    Score A0   Coverage A0
              Coverage   peptides

gi3 8491705    13.54        12         9.22        13.54
gi73913089     14.42        15         6.27        14.42
gi58374258      1.5          5         5.63         1.5
gil83197180     3.47         3         5.27        3.47
gi255984636     7.5          4         4.80         7.5
gi256012108    14.04         3         4.53        14.04
gi9756252       4.01         2         4.46        4.01
gi67082579     10.47         2         4.43        10.47
gi2290009       7.16        11         4.33        5.87
gil67886806      56          4         4.29         56
gi23344577     12.12         5         4.36        12.12
gi4324808       2.51         7         4.05         1.6
gi222533599    23.29         4         3.85        23.29
gi71060450      4.85         4         3.84        4.85
gi37935985     11.76         4         3.84        11.76
gil08860432     3.33         2         3.74        3.33
gill4801226     5.26         2         3.70        5.26
gi22596451      3.81         4         3.68        3.81
gil83200570     3.51         2         3.68        3.51
gi34786230      10.8         3         3.60        10.8
gil002239      18.27         2         3.54        18.27
gi77168129     13.68         3         3.40        13.68
gi222532593     8.53         3         2.61        8.53
gi219688191     9.09         6         2.56        9.09
gi255687141     9.03         1         2.52        9.03
gi222532161     9.09         6         2.45        9.09
gi37934078      2.79         2         2.42        2.79
gi405003       12.08         1         2.41        12.08
gi54792352      9.39         2         2.34        9.39
gi3885826      11.36         2         2.31        11.36

Accession     # peptides
                  A0

gi3 8491705       4
gi73913089        4
gi58374258        3
gil83197180       3
gi255984636       2
gi256012108       2
gi9756252         2
gi67082579        2
gi2290009         3
gil67886806       2
gi23344577        2
gi4324808         2
gi222533599       2
gi71060450        2
gi37935985        2
gil08860432       2
gill4801226       2
gi22596451        2
gil83200570       2
gi34786230        3
gil002239         2
gi77168129        2
gi222532593       1
gi219688191       1
gi255687141       1
gi222532161       1
gi37934078        2
gi405003          1
gi54792352        2
gi3885826         1

Accession # = NCBI NR database, #AAs = total number of amino acids in
the protein entry, MW = molecular weight of the protein in kDa,
Description: description from the NCBI database, and Peptides = total
number of unique peptides found.

Table 3: Presence of HIV-1 proteins in HIV+ patient urinary EVs.

ID      ART   AIDS   Viral load    CD4 cells/ul   Nef   Gag   Pol
                      copies/ml

22      No                             224         X     X     X
27      Yes   AIDS       <50           134         X
28      Yes   AIDS     280100           22         X     X
30      Yes   AIDS     >10000          <20         X     X     X
41      Yes             29187          440         X     X     X
46      Yes              <50           689               X     X
45      Yes              400           345         X
48      Yes             4974           454         X
51                       NA             NA         X     X     X
52      Yes              51            574         X     X     X
61                       <50           655               X     X
62      Yes   AIDS       <50           232         X
63      No              2023            83         X
65      No               NA             NA
66      No               NA             NA                     X
67      Yes              75            509         X
68      No               NA             NA
69      Yes              <50           187         X
70      Yes              <50           399         X
71      Yes              <50           456
74                       NA             NA
86      Yes              <75           1642        X     X     X
103     Yes   AIDS       150           560         X     X     X
104     Yes   AIDS       77            313         X     X
108     Yes   AIDS       <50           653         X     X     X
110     Yes              <50           379         X     X
111     Yes   AIDS       <50           182               X     X
112     Yes   AIDS      >200           581                     X
142     Yes              <50           487         X     X     X
173-1   Yes              <50           398         X     X     X
173-2   Yes              <50           398         X     X     X
174-1   Yes              48            315         X     X     X
174-2   Yes              48            315         X     X     X
196-1   Yes   AIDS       <50           113         X     X     X
196-2   Yes   AIDS       <50           113         X     X     X

ID      Protease   Rev   RT   Tat   Vif   p1   p24   p17   Poly

22         X        X          X     X    X     X     X
27
28         X             X           X
30         X             X     X     X          X     X     X
41
46                  X    X
45         X        X
48                  X          X     X
51         X             X
52                  X          X
61
62
63
65
66
67
68
69
70
71
74
86         X
103
104
108
110
111
112                      X
142                 X          X                            X
173-1      X        X    X     X     X          X
173-2      X             X           X
174-1               X    X           X
174-2                                X
196-1      X        X    X     X     X          X     X     X
196-2      X        X    X     X     X          X     X     X

ID      Vpu   Env   Vpr   Vif

22
27
28
30
41
46
45             X           X
48
51
52       X           X     X
61
62
63
65             X
66
67
68             X
69
70
71             X
74             X
86
103
104
108
110
111
112
142      X                 X
173-1    X
173-2
174-1
174-2
196-1    X                 X
196-2    X                 X

An initial protein identification list was generated from matches
with an Xcorr score versus charge state of 1.0 (+1), 1.5 (+2), and
1.7 (+3) and consensus scores greater than 10.0; NA = not available.

Table 4: Exosomal proteins found in urinary EVs from E1IV+ patients.

           Genes in our    Genes in the    Percentage      Fold
             analysis        FunRich        of genes     enrichment
                             database

Exosomal       1932            2001           29.44         2.11
proteins

AIBG, A2M, AARS, ABCA7, ABCBl, ABCB11, ABCB6, ABCC1, ABCC11, ABCC9,
ABCG2, ABHD8, ACAA2, ACATI, ACAT2, ACE, ACE2, ACLY, AC01, ACOT11,
ACP2, ACSL3, ACSL4, ACSM1, ACTAl, ACTA2, ACTB ACTO, ACTGl ACTL6A,
ACTNl, ACTN2, ACTN4, ACTR1A, ACTR1B, ACTR2, ACTR3, ACY1, ACY3,
ADAM10, ADAMTS3, ADCYl, ADH5, ADH6, ADK, ADSL, AEBP1, AGAP2, AGR2,
AGR3, AGRN, AGT, AHCTF1, AHCY AHNAK, AHSA1, AHSG, AK1, AK2, AKAP9,
AKR1A1, AKR1B10, ALAD, ALB, ALCAM, ALDH1OA1, ALDH1A1, ALDH1L1,
ALDH2, ALDH3B1, ALDH8A1, ALDOA, ALDOB, ALDOC, ALK, ALOX12, ALPL,
ALPP, ALYREF, AMN, ANGPT1, ANGPTL1, ANGPTL4, ANKFY1, ANKRD11, ANOI,
AN06, ANPEP, ANXAI, ANXA11, ANXA13, ANXA3, ANXA4, ANXA6, ANXA7,
AOX1, AP1M1, AP2A1, AP2A2, AP2M1, AP4M1, A PA FI, APLP2, APOA APOB,
APOD, APOE, APOL1, APP, APPLI, APPL2, APRT, AQP2, ARF5, ARFIP1,
ARHGAP1, ARHGAP23, ARHGDIA, ARHGDIB, ARHGEF12, ARHGEF18, ARL15,
ARL3, ARL8B, ARMC3, ARMC9, ARPC1A, ARPC1B, AR ARPC3, ARPC5, ARRDC1,
ARSE, ARSF, ARVCF, ASAH1, ASB6, ASL, ASNA1, ASNS, ATAD2, ATIC, AT
PIAL ATP1A2, ATP1A3, ATP2B1, ATP2B2, ATP2B4, ATP4A, ATP5A1, ATP5B,
ATP5L, ATP6AP1, ATP6AP2, ATP6V0A4, ATP6V0C, ATP6V0D1, ATP6V0D2,
ATP6V1A, ATP6VLB 1, ATPOV1C1, ATP6V1C2, ATP6V1D, ATPOV1E1, ATP6V1H,
ATRN, AUP1, AZGP1, AZU1, B2M, B3GAT3, B4GALT1, B4GALT3, BAIAP2,
BAIAP2L1 BCAM, BCR, BDH2, BGN, BHLHB9, BHMT, BHMT2, BLMH, BLOC1S5,
BLVRA, BLVRB, BMP3, BPI, BPIFB1, BPTF, BRI3BP, BROX, BSG, BTG2,
BTN1A1, Cllorf52, Cllorf54, C16orf80, C16orf89, C17orf80, Clorfllo,
C1QC, C1QTNF1, C1QTNF3, C1R, C2orfl6, C3, C4BPA, C5, C9, CAB39L,
CACNA2D1, CACYBP, CAD, CALMI, CALML3, CALR, CAMK4, CAMP, CANDI,
CANX, CAPI, CAPN1, CAPN2, CAPN5, CAPN7, CAPZA2, CAPZB, CARD11,
CASP9, CAVI, CBR3, CC2D1A, CCDC105, CCDC132, CCL28, CCPG1, CCT2,
CCT3, CCT4, CCT5, CCT6A, CCT7, CCT8, CD101, CD14, CD163L1, CD19,
CD2, CD22, CD274, CD2AP, C CD40, CD44, CD53, CD55, CD58, CD59,
CD63, CD70, CD74, CD79B, CD80, CD81 CD9, CD97, CDC42, CDC42BPA,
CDC42BPB, CDH1, CDH17, CDHR2, CDHR5, CDK1, CDK5RAP2, CDKL1,
CEACAM5, CELSR2, CE CES2, CETP, CFD, CFH, CFI, CELI, CHGB, CHID1,
CHMP1A, CHMP2B, CHMP4B, CHRDL2, CHST1, CHST14, CIBI, CKAP4, CKB,
CLASP1, CLCA4, CLDN3, CLDN4, CLDN7, CLIC1, CLIC4, CLIC5, CLIC6,
CLIP CLTCL1, CLU, CMPK1, CNDP2, CNKSR2, CNTLN, COASY, COBLL1,
COL12A1, COL15A1, COL18A1, COL6A1, COL6A2, COL6A3, COLEC10,
COLGALT1, COMT, COPA, COPB1, COPB2, COPS8, COROIA, COROIB, COX
COX5B, CP, CPD, CPN2, CPNE1, CPNE3, CPNE5, CPNE8, CPVL, CRI, CR2,
CRB2, CREB5, CRISPLD1, CRNN, CRTC2, CRYAB, CRYZ, CS, CSE1L, CSK,
CSPG4, CSRP1, CST4, CSTB, CTDSPL, CTNNA1, CTNNB1 CTSC, CTSG, CTTN,
CUBN, CUL3, CUL4B, CUTA, CUX2, CXCR4, CYB5R1, CYBRD1, CYFIP1,
CYFIP2, CYP2J2, DAAM2, DAG1, DAK, DARS, DBNL, DCD, DCTN2, DCXR,
DDAH1, DDAH2, DDB1, DDC, DDR1, DDX1 DDX19B, DDX21, DDX23, DDX3X,
DDX5, DERA, DHCR7, DHX34, DHX36, DHX9, DIAPH2, DIP2A, DIP2B, DIP2C,
DLD, DLG1, DMBT1, DNAH7, DNAH8, DNAJA1, DNAJA2, DNAJB1, DNAJB9,
DNAJC13, DNAJC3, D DNHD1, DNM2, DNPH1, DOCKIO, DOCK2, DOPEY2,
DPEP1, DPP3, DPP4, DPYS, DPYSL2, DSC2, DSG2, DSG3, DSP, DSTN,
DUOX2, DUSP26, DUT, DYNC1H1, DYNC2H1, DYSF, ECE1, ECHI, ECM1,
EDIL3, EEA1, EEF1D, EEF1G, EEF2, EFEMP1, EFEMP2, EGF, EGFR, EHD1,
EHD2, EHD3, EHD4, EIF2S1, EIF2S3, EIF3A, EIF3B, EIF3E, EIF3L,
EIF4A1, EIF4A2, EIF4A3, EIF4E, EIF4G1, EIF4H, ELANE, EML5, ENOL
ENPP6, ENTPD1, EPB41L2, EPCAM, EPHA2, EPHA5, EPHB1, EPHB2, EPHB3,
EPHB4, EPHX2, EPN3, EPPK1, EPRS, EPS8, EPS8L1, EPS8L2, EPS8L3, ERA
PI, ERBB2, ERMN, EROIL, ERP44, ESD, ET FA, EVP EXT2, EYS, EZR, Fll,
F11R, F5, F7, FAB PI, FABP3, FAH, FAM129A, FAM129B, FAM151A,
FAM208B, FAM209A, FAM20A, FAM20C, FAM49B, FAM65A, FAS, FASLG, FASN,
FATI, FAT2, FBL, FBP1, FBP2, FGA, FGB, FGG, FGL2, FGR, FH, FIGNL1,
FKBP1A, FKBP4, FKBP5, FLNA, FLNB, FLNC, FLOT1, FLOT2, FMNL1, FN1,
FOLH1, FRK, FSCN1, FTCD, FUCA1, FURIN, FUS, FUT2, FUT3, FUT6, FUT8,
FUZ, G6 GAL3ST4, GALK1, GALM, GALNT3, GANAB, GARS, GART, GATSL3,
GBE1, GBP6, GCN1L1, GCNT2, GCNT3, GDF2, GDI2, GDPD3, GEMIN4, GFPT1,
GGCT, GGH, GGT1, GHITM, GIPC1, GK, GK2, GLB1, GLDC, GL GLOl, GLUD1,
GLUL, GNA13, GNAU, GNAI2, GNAQ, GNAS, GNBL GNB2, GNB2L1, GNB3,
GNB4, GNB5, GNG12, GNPDA1, GNPTG, GOLGA4, GOLGA7, GOTI, GOT2, GPC1,
GPC4, GPD1, GPI, GPM6A, GPR155, GPR GPRASP1, GPRC5A, GPRC5B, GPT,
GREB1, GRHPR, GRIDI, GRIN1, GRK4, GSN, GSR, GSS, GSTA3, GSTCD,
GSTK1, GSTOl, GSTP1, GUSB, H1FOO, H2AFY, H2AFY2, HADHA, HAPLN3,
HAUS5, HBB, HBD, HBS1L HEBP1, HEBP2, HEPH, HGD, HGS, HINT1, HIRA,
HIST1H1B, HIST1H2BA, HIST1H2BL, HIST2H2AC, HLA-A, HLA-B, HLA-DPB1,
HLA-DQB1, HLA-DRB1, HLA-DRB5, HLAE, HNMT, HNRNPA1, HNRNPA2B1,
HNRNPC, HNRNPF, HNRNPK, HNRNPL, HP, HPD, HPGD, HPR, HPRT1, HRG,
HRNR, HSD17B10, HSD17B4, HSP90ABL HSP90BL HSPA12A, HSPA13, HSPA1L,
HSPA2, HSPA4, HSPA5, HSPA6, HSPA8, HSPA9, HSPB1, HSPB8, HSPG2,
HSPH1, HTATIP2, HTRA1, HUWE1, HYOU1, IARS, ICAM1, ICAM3, IDH1,
IFITM2, IFITM3, IGF2R, IGFALS, IGSF3, IGSF8, IKZF5, IMPDH2, INADL,
INSR, IQCB1, IQCG, IQGAP1, IQGAP2, IRF6, I ITGA2, ITGA2B, ITGA3,
ITGA4, ITGA6, ITGAL, ITGAV, ITGBL ITGB2, ITGB3, ITGB4, ITGB7,
ITGB8, ITIH2, ITIH4, ITM2C, ITSN1, ITSN2, IVL, JADE2, JUP, KALRN,
KCNG2, KHK, KIAA1324, KIF12, KIF9, KIFC3, KL, KNG1, KPNBL KPRP,
KRT1, KRT10, KRT12, KRT14, KRT15, KRT16, KRT17, KRT18, KRT19, KRT2,
KRT20, KRT24, KRT25, KRT27, KRT28, KRT3, KRT5, KRT6C, KRT7, KRT73,
KRT75, KR KRT8, KRT9, L1CAM, LAD1, LAMA3, LAM A4, LAMA5, LAMB2,
LAMB 3, LA MCI, LAMC2, LAMPI, LAMP2, LAMTOR3, LBP, LCK, LCP1, LDHA,
LDHB, LEPRE1, LFNG, LGALS3, LGALS3BP, LGALS4, LIMAI, LIN7 LMAN1,
LMAN2, LOXL4, LPO, LRP1, LRP1B, LRP2, LRP4, LRPPRC, LRRC15,
LRRC16A, LRRC57, LRRK2, LRSAM1, LSP1, LSR, LTA4H, LTBP3, LTF,
LUZP1, LYPLA2, MAGI3, MAL2, MANI Al, MAN1A2, MAN2A MARCKSLl, MARK3,
MARS, MARVELD2, MASPl, MASP2, MBD5, MBLAC2, MCAM, MCPH1, MDH1,
MDH2, MEGF8, MEP1A, MEST, METRNL, MFGE8, MFI2, MGAM, MG ATI, MG AT
4 A, MID2, MIF, MINK1, MLLT3, ML MME, MMP24, MMP25, MMRN1, MMRN2,
MNDA, MOB1A, MOB1B, MOGS, MPO, MPP5, MPP6, MS4A1, MSH6, MSN, MSRA,
MTA1, MTAP, MTCH2, MTHFD1, MTMR11, MTMR2, MUC13, MU Cl 6, MUC4,
MUM1L1, MVB12A, MVB12B, MVP, MX1, MXRA5, MXRA8, MYADM, MYH10,
MYH11, MYH13, MYH14, MYH3, MYH8, MYH9, MYL6B, MY015A, MYOIB, MYOIC,
MYOID, MYOIE, MYOIG, MY05B, MY06, MYOF, N4BP2L2, NAA16, NAA50, NA
NAGLU, NAMPT, NAP1L4, NAPA, NAPG, NAPRT, NAPSA, NARS, NBR1, NCALD,
NCCRP1, NCKAP1, NCKAP1L, NCL, NCOA3, NCSTN, NDRG1, NDRG2, NEB,
NEBL, NEDD4, NEDD4L, NEDD8, NEU1, NIDI, NIN, NIPB NKX61, NONO,
NOTCH1, NOX3, NPC1, NPEPPS, NPHS1, NPHS2, NPM1, NPNT, NQ02, NT5C,
NT5E, NUCB1, NUCB2, NUDT5, NUMA1, NXPE4, OLA1, OPTN, OR2A4, OS9,
OSBPL1A, OXSR1, P2RX4, P4HB, PA2G4, PACSIN3, PADI2, PAFAH1B1,
PAFAH1B2, PAGE2, PAICS, PAM, PARD6B, PARP4, PBLD, PCBP1, PCDHGB5,
PCK1, POLO, PCNA, PCSK9, PCYOX1, PDCD2, PDCD5, PDCD6, PDCD6IP,
PDDC1, PDE8A, PDIA2, PDI PDIA6, PDLIM7, PDZK1, PEBP1, PECAM1, PEF1,
PEPD, PEX1, PFAS, PFKL, PFKP, PGAM1, PCD, PGKL PGLYRP1, PGM1, PHB2,
PHGDH, PI4KA, PIGR, PIK3C2A, PIK3C2B, PILRA, PIP, PIP4K2C, PKD1,
PKD PKLR, PKM, PKN2, PKP3, PLAT, PLAU, PLCB1, PLCD1, PLCG2, PLD3,
PLEC, PLEKHA1, PLEKHA7, PLEKHB2, PLG, PLIN2, PLOD1, PLOD2, PLOD3,
PLS1, PLSCR1, PLTP, PLVAP, PLXNA1, PLXNB2, PM20D1, POFUT2, PONI,
PON3, POTEE, POTEF, POTEI, POTEM, PPA1, PPARG, PPFIA2, PRIA, PPIB,
PPL, PPM1L, PPP1CB, PPP1R7, PPP2CA, PPP2R1A, PPP2R1B, PROP, PRDXL
PRDX3, PRDX4, PRDX5, PRG4, PRKAR PRKCH, PRKCI, PRKCZ, PRKDC,
PRKRIP1, PRNP, PROM1, PROM2, PROSI, PROZ, PRRC2A, PRSS23, PRTN3,
PSAP, PSAT1, PSMA2, PSMA3, PSMA5, PSMA7, PSMB1, PSMB3, PSMB4,
PSMB5, PSMB6, PSMB8, PSM PSMC4, PSMC6, PSMD11, PSMD12, PSMD13,
PSMD2, PSME1, PSME2, PSME3, PTBP1, PTER, PTGFRN, PTGR1, PTGS1,
PTPN13, PTPN23, PTPRA, PTPRC, PTPRF, PTPRJ, PTPRO, PTRF, PTX3,
PYGB, PYGL, QDP QSOX1, RABIO, RAB11B, RAB17, RABIA, RAB1B, RAB22A,
RAB25, RAB29, RAB2A, RAB34, RAB3B, RAB3GAP1, RAB43, RAB4B, RAB6B,
RAB7A, RAB8A, RAB8B, RAB9A, RAC1, RACGAP1, RALA, RALB, RAP1A,
RAP2A, RAPGEF3, RARRES1, RARS, RASAL3, RASSF9, RBL2, RCC2, REG4,
RELN, RENBP, RFC1, RFTN1, RHEB, RHOB, RHOF, RIMS2, RLF, RNASE7,
RNF213, RNH1, RNPEP, R0B02, ROCK2, RP2, RPL10, RPL RPL23, RPL3,
RPL30, RPL34, RPL35A, RPL4, RPL5, RPL6, RPL8, RPLP2, RPN1, RPS11,
RPS14, RPS15A, RPS16, RPS18, RPS2, RPS20, RPS21, RPS27A, RPS3A,
RPS4X, RPS4Y1, RPS4Y2, RPS7, RPS9, R RUVBL1, RUVBL2, RYR1, S100A11,
S100A6, S100P, SAA1, SAFB2, SAMM50, SARS, SBSN, SCAMP2, SCAMP3,
SCARB1, SCARB2, SCEL, SCIN, SCN10A, SCN11A, SCPEP1, SCRIB, SCRN2,
SDCBP, SDF4, SEC31 SEMA3G, SEPPI, SERB PI, SERINC1, SERINC2,
SERINC5, SERPINA1, SERPINA3, SERPINA4, SERPINA5, SERPINA7,
SERPINB1, SERPINB13, SERPINB6, SERPINB9, SERPING1, SETD4, SFI1,
SFN, SFRP1, SF SHMT2, SHROOM2, SIAE, SIRPA, SITI, SLAM FI, SLAMF6,
SLC12A1, SLC12A2, SLC12A3, SLC12A7, SLC12A9, SLC13A2, SLC13A3,
SLC15A2, SLC16AL SLC1A1, SLC1A4, SLC1A5, SLC20A2, SLC22A11, SLC2
SLC22A5, SLC22A6, SLC23A1, SLC25A1, SLC25A3, SLC25A4, SLC25A6,
SLC26A11, SLC26A4, SLC26A9, SLC27A2, SLC29A1, SLC2A1, SLC2A3,
SLC34A2, SLC35D1, SLC36A2, SLC37A2, SLC38A1, SLC39A5, SLC44A2,
SLC44A4, SLC46A3, SLC4A1, SLC4A4, SLC5A1, SLC5A10, SLC5A2, SLC5A5,
SLC5A6, SLC5A8, SLC5A9, SLC6A13, SLC6A14, SLC6A19, SLC7A5, SLC9A1,
SLC9A3, SLC9A3R1, SLC9A3R2, SLC04C1, SMIM22, SMIM24, SMO, SMPDL3B,
SMURF1, SNCG, SND1, SNRNP200, SNX12, SNX18, SNX25, SNX33, SNX9,
SODI, SO GAI, SORD, SORL1, SORTI, SPAG9, SPAST, SPEN, SPINK1,
SPON2, SPRR3, SPTAN1, S SRPR, SRSF7, ST13, ST3GAL1, ST3GAL6,
STAMBP, STAU1, STIPI, STK10, STK11, STK24, STOM, STRIP1, STX3,
STX4, STX7, STXBP1, STXBP2, STXBP3, STXBP4, SUBI, SUCLA2, SUSD2,
SYAP1, SYNE1, TAF6L, TALDOl, TAOK1, TARS, TAX1BP1, TAX1BP3,
TBC1D10A, TBC1D21, TC2N, TCP1, TECTA, TEKT3, TEX14, TF, TFRC,
TGFB1, TGFBI, TGFBR3, TGM1, TGM2, TGM3, TGM4, THBS1, THBS2, THRAP3,
THS TINA GLI, TJP2, TKT, TLN1, TLR2, TM7SF3, TM9SF2, TMBIM1, TMC6,
TMC8, TMED2, TMED9, TMEM109, TMEM192, TMEM2, TMEM256, TMEM27,
TMEM63A, TMPRSS11B, TMPRSS11D, TMPRSS2, TNFAIP3, TNFRS TNFSF10,
TNFSF13, TNIK, TNKS1BP1, TNP03, TOLLIP, TOMI, TOM1L2, TOMM70A,
TOR1A, TO RIB, TOR3A, TPI1, TPM 3, TPP1, TPRG1L, TRAP1, TREH,
TRIP10, TSNAXIP1, TSPAN1, TSPAN15, TSPAN3, TS TTC18, TTLL3, TTN,
TTR, TUBA IB, TUBA4A, TUBB3, TUBB4A, TUBBS, TUFM, TWF2, TXNDC16,
TXNDC8, TXNRD1, TYK2, TYRP1, UACA, UBAI, UBACI, UBASH3A, UBE2N,
UBE2V2, UBL3, UBXN6, UEVLD, UGD UGGT1, UGP2, ULK3, UMOD, UPB1,
UPK1A, UPK3A, UQCRC2, UTRN, UXS1, VAMPI, VA MP3, VAMP7, VAPA, VASN,
VASP, VATI, VCL, VCP, VDAC3, VILI, VIM, VMOl, VPS13C, VPS13D,
VPS28, VPS35, VPS3 VPS37D, VPS4A, VPS4B, VTA1, VWA2, VWF, WARS,
WAS, WASF2, WASL, WDR1, WIZ, WNT5B, XDH, XPNPEP2, XPOl, XRCC5,
XRCC6, YBX1, YES1, YWHAE, YWHAG, YWHAH, YWHAZ, ZCCHC11, ZDHHC1,
ZFYVE20, ZG16B, ZMPSTE24, ZNF114, ZNF486, ZNF571, and ZNHIT6.

GENE: ExoCarta (http://exocarta.org/exosome_markers_new) [33];
GENE: EV antibody array [35]; GENE: HIV exosomal proteins [36].

Table 5: Exosomal proteins found in urinary EVs from uninfected
controls.

             Genes in      Genes in the    Percentage      Fold
            our analysis     FunRich        of genes     enrichment
                             database

Exosomal         37            2001           72.54         5.26
proteins

A1BG, ACTA1, ACTA2, ACTB, ACTBL2, ACTC1, ACTG1, ACTG2, ALB, AMBP,
APOA1, APOD, AZGP1, B2M, CDH1, CLU, CP, CRNN, DCTN2, EGF, HP, HPR,
HSPB1, ITIH4, KNG1, LAMA3, LMAN2, POTEE, POTEF, POTEI, S100A8,
SERPINA1, SERPING1, TF, TTR, UMOD, and VASN.

Table 6: Functional enrichment analysis of HIV+ EV proteins.

                                 Genes in the    Genes in the
                                    dataset      Bkg. database

Molecular function
  Protein serine/threonine            272            5,602
    kinase activity
  Catalytic activity                  456             827
  GTPase activator activity           131             836
  Guanyl-nucleoside exchange          105             614
    factor activity
  Cell adhesion molecule              307             531
    activity
Biological process
  Regulation of nucleobase,          2,236           4,658
  nucleoside, and
  nucleic acid
Biological pathway
  Integrin cell surface               69             1,366
  interactions

                                 Percentage      Fold
                                  of genes     enrichment

Molecular function
  Protein serine/threonine           30           1.18
    kinase activity
  Catalytic activity                 4.9          1.1
  GTPase activator activity          4.7          1.2
  Guanyl-nucleoside exchange         3.6          1.2
    factor activity
  Cell adhesion molecule             3.3          1.1
    activity
Biological process
  Regulation of nucleobase,         24.8          1.05
  nucleoside, and
  nucleic acid
Biological pathway
  Integrin cell surface             23.3          1.2
  interactions

                                  Corrected P
                                 value (BH FDR)

Molecular function
  Protein serine/threonine          1.04-08
    kinase activity
  Catalytic activity                1.12-05
  GTPase activator activity         8.14-05
  Guanyl-nucleoside exchange        8.54-05
    factor activity
  Cell adhesion molecule             0.0001
    activity
Biological process
  Regulation of nucleobase,         3.24-05
  nucleoside, and
  nucleic acid
Biological pathway
  Integrin cell surface               0.03
  Interactions

Table 7: Comparison of pathways between HIV+ groups from Pathway
Studio 11.4.

HIV group               Pathway

CD4+ T cells greater    Natural killer cell inhibitor receptor
than 300 n = 15           signaling
                        Intermediate filament polymerization
                        Ca2+ flux regulation
                        G1/S phase transition
                        G2/M phase transition
                        S/G2 phase transition
                        Protein folding
                        Golgi to endosome transport
                        Endosomal recycling
                        Kinetochore assembly

CD4+ T cells less       Neutrophil chemotaxis
than 300 n = 15         Vascular motility
                        Platelet activation via GPCR signaling
                        Insulin influence on protein synthesis
                        mTOR signaling overview
                        EDNRA/B [right arrow] vascular motility
                        Proplatelet maturation
                        Natural killer cell activation through
                          ITAM-containing receptors
                        Taste sensor receptors activates
                          mTOR signaling
                        Natural killer cell activation

Low VLs n = 14          Intermediate filament polymerization
                        Natural killer cell inhibitory receptor
                          signaling golgi to endosome transport
                        golgi to endosome transport
                        Ca+ fux regulation
                        HRH1/3 [right arrow] synaptic transmission
                        Vascular motility
                        Endosomal recycling
                        G1/S phase transition
                        Golgi transport
                        G2/M phase transition

High VLs n = 10         Metaphase/anaphase phase transition
                        S/G2 phase transition
                        Spindle assembly
                        Natural killer cell activation
                        Histone ubiquitylation
                        Eosinophil survival by cytokine signaling
                        Protein folding
                        G2/M phase transition

Table 8: Functional enrichment analysis of control EV proteins.

                         Genes in the    Genes in the    Percentage
                           database      Bkg. database    of genes

Site of expression
  Cervicovaginal fluid        16              544           12.0
  Neutrophils                 13              392            9.7
  Gastric juice                9              222            6.7
Molecular function
  Defense/immunity             5              52             3.7
  protein activity
Biological process
  Immune response             13              561            9.8
  Signal transduction         43             3907           32.5
  Cell communication          41             3687           31.1
  Antigen presentation         1               1             0.7

                           Fold       Corrected
                         enrichment   P value
                                      (BH FDR)

Site of expression
  Cervicovaginal fluid      4.2       2.59E-06
  Neutrophils               4.8       6.68E--06
  Gastric juice             6.1        4E--05
Molecular function
  Defense/immunity          15.7      3.96E-05
  protein activity
Biological process
  Immune response           3.4        0.00026
  Signal transduction       1.5        0.0026
  Cell communication        1.5        0.0028
  Antigen presentation     134.4       0.0073

Table 9: Overlapping EV proteins from HIV+ and HIV- samples,
LC/MS/MS analysis.

Gene

ABCB1      ATP-binding cassette, subfamily B (MDR/TAP), member 1
ANXA8      Annexin A8
ASIC1      Acid-sensing (proton-gated) ion channel 1
ASIC2      Acid-sensing (proton-gated) ion channel 2
AUTS2      Autism susceptibility candidate 2
AZU1       Azurocidin 1
BCAT1      Branched chain amino acid transaminase 1, cytosolic
BRD4       Bromodomain containing 4
CCL5       Chemokine (C-C motif) ligand 5
CEACAM8    Carcinoembryonic antigen-related cell adhesion molecule 8
CFH        Complement factor H
CHIT1      Chitinase 1 (chitotriosidase)
CLDN7      Claudin 7
COL16A1    Collagen, type XVI, alpha 1
CPB2       Carboxypeptidase B2 (plasma)
CRADD      CASP2 and RIPK1 domain containing adaptor with death
           domain
CTSG       Cathepsin G
CYP4A11    Cytochrome P450, family 4, subfamily A, polypeptide 11
DEFA1      Defensin, alpha 1
DNAH17     Dynein, axonemal, heavy chain 17
DUSP9      Dual specificity phosphatase 9
EIF4A1     Eukaryotic translation initiation factor 4A1
ELANE      Elastase, neutrophil expressed v-erb-b2 avian
           erythroblastic leukemia viral oncogene homolog 2
FARP1      FERM, RhoGEF (ARHGEF), and pleckstrin domain protein 1
           (chondrocyte-derived)
GDF15      Growth differentiation factor 15
GNA12      Guanine nucleotide binding protein (G protein) alpha 12
GNL1       Guanine nucleotide binding protein-like 1
GRIN2A     Glutamate receptor, ionotropic, N-methyl D-aspartate 2A
HAAO       3-Hydroxyanthranilate 3,4-dioxygenase
HAL        Histidine ammonia-lyase
HBA1       Hemoglobin, alpha 1
HBB        Hemoglobin, beta
HBD        Hemoglobin, delta
IGKC       Immunoglobulin kappa constant
LGALS3     Lectin, galactoside-binding, soluble, 3
MEF2C      Myocyte enhancer factor 2C
MLLT4      Myeloid/lymphoid or mixed-lineage leukemia
           (trithorax homolog, Drosophila); translocated to, 4
MPO        Myeloperoxidase
MRC2       Mannose receptor, C type 2
MYBPC3     Myosin binding protein C, cardiac
NCAM1      Neural cell adhesion molecule 1
NKTR       Natural killer-tumor recognition sequence
NUP93      Nucleoporin 93 kDa
PDE1C      Phosphodiesterase 1C, calmodulin-dependent 70 kDa
PDLIM5     PDZ and LIM domain 5
PIK3R1     Phosphoinositide-3-kinase, regulatory subunit 1 (alpha)
RAB31      RAB31, member RAS oncogene family
RAP1GAP    RAP1 GTPase activating protein
REG1A      Regenerating islet-derived 1 alpha
RNASE2     Ribonuclease, RNase A family, 2
           (liver, eosinophil-derived neurotoxin)
RNASE3     Ribonuclease, RNase A family, 3
RPS14      Ribosomal protein S14
RUNX2      Runt-related transcription factor 2
SHBG       Sex hormone-binding globulin
SLC22A5    Solute carrier family 22 (organic cation/carnitine
           transporter), member 5
SLC6A6     Solute carrier family 6 (neurotransmitter transporter),
           member 6
TACC2      Transforming, acidic coiled-coil containing protein 2
TAF6L      TAF6-like RNA polymerase II, p300/CBP-associated factor
           (PCAF)-associated factor, 65 kDa
TNIK       TRAF2 and NCK interacting kinase
TRAPPC12   Trafficking protein particle complex 12
TRIM58     Tripartite motif containing 58
WNT2B      Wingless-type MMTV integration site family, member 2B
WNT6       Wingless-type MMTV integration site family, member 6

Table 10: Functional analysis of overlapping HIV+ and HIV-
EV proteins.

                             Genes in the   Genes in the
                               data set     Bkg. database

Site of expression
  Urine                           31            3202
  Cervicovaginal fluid            12             544
  Neutrophils                     9              392
  032403_BALF4_glypep             4              43
  Neutrophil                      19            1979
  Monocyte                        23            2786
Cellular component
  Extracellular                   22            1808
  Stored secretory granule        3              19
  Lysosome                        17            1609
  Extracellular space             8              399
  Exosomes                        19            2001
  Azurophil granule               2               6

                             Percentage     Fold
                              of genes    enrichment

Site of expression
  Urine                         51.7         3.0
  Cervicovaginal fluid          20.0         7.2
  Neutrophils                   15.0         7.7
  032403_BALF4_glypep           6.7          35.0
  Neutrophil                    31.7         3.0
  Monocyte                      38.3         2.6
Cellular component
  Extracellular                 37.9         3.1
  Stored secretory granule      5.2          51.0
  Lysosome                      29.3         2.8
  Extracellular space           13.8         5.6
  Exosomes                      32.8         2.5
  Azurophil granule             3.4         108.9

                                 Corrected
                                  P value
                             (Bonferroni method)

Site of expression
  Urine                           6.85E--07
  Cervicovaginal fluid            5.33E-05
  Neutrophils                     1.56E-03
  032403_BALF4_glypep             3.53E-03
  Neutrophil                      3.67E--03
  Monocyte                        3.90E-03
Cellular component
  Extracellular                   4.61E-05
  Stored secretory granule        3.27E--03
  Lysosome                        7.73E-03
  Extracellular space             1.05E-02
  Exosomes                        1.14E-02
  Azurophil granule               1.35E-02

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.

Site of expression

Endothelium   75.4   **
Plasm         67.9   **
Liver         65.5   **
Kidney        57.8   **
Lung          56.3   **
Seruml        44.1   **

Cellular component

Cytoplasm    32.1   **
Nucleus      32.4   **
Plasma mem   19.3   **
Exosomel     11.2   **
Lysosomel     8.9   **

Note: Table made from bar graph.
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Article Details
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Title Annotation:Research Article
Author:Anyanwu, Samuel I.; Doherty, Akins; Powell, Michael D.; Obialo, Chamberlain; Huang, Ming B.; Quarshi
Publication:Advances in Virology
Article Type:Report
Geographic Code:1USA
Date:Jan 1, 2018
Words:11120
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