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

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.


Introduction
Extracellular vesicles (EVs) are membrane bound vesicles, between 30 nm and 1 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][9][10][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][13][14][15][16]. The glomerular capsule filters blood that is passed into the renal tubule and accounts for thirty percent of the urinary protein content [14][15][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][19][20][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][25][26][27][28][29][30]. The objectives of this study were to determine the differences in proteins from urinary EVs from HIV+ patients and HIV− individuals 2 Advances in Virology 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.

Methods
. . 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.
. . EV Isolation. Urine samples were centrifuged at 1000 ×g to remove cells and sediment then frozen at −80 ∘ 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 ×g for 15 minutes at 4 ∘ C. The retentate, containing EVs, was collected from the top of the filter and resuspended in 200 l phosphate buffered saline (PBS) for use in the transmission electron microscopy and tandem mass spectrometry (LC/MS/MS) analysis.
. . 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 ∘ 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 ∘ 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).
. . Nanoparticle Tracking Analysis (NTA). Urine samples from HIV-negative ( = 8) and positive individuals ( = 11), 15 ml, were centrifuged at 300 ×g for 10 min at 4 ∘ C to remove cell debris. The supernatant was collected and centrifuged at 16,500 ×g for 20 min at 4 ∘ C and the supernatant collected and ultracentrifuged at 120,000 ×g at 4 ∘ C for 1.5 hr. The pellet was resuspended in 500 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 ∘ 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.
. . 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 ∘ 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/ l for 4 hours at 37 ∘ 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 × 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 Fun-Rich (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 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 ( = 15) to those with less than 500 ( = 15) and HIV high VL, greater than 200 copies ( = 10), compared to HIV low viral loads, less than 200 copies ( = 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.
. . 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 ∘ 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.

. . 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 ( = 35) contained at least one HIV-1 protein in EVs, while no HIV proteins were found in the HIV− samples ( = 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.
. . 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 ± 0.0733 and 3.69 ± 0.075, respectively ( < 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.
. . 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 ( < 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 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. Yes 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.
Advances in Virology     Functional analysis of the control EVs are listed in Table 8. The major sites of expression were cervicovaginal fluid, neutrophils, and gastric juice ( < 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 ( < 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 ( < 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.

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][39][40][41][42][43][44][45][46][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  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 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-and IL1. Both these cytokines were present in the EVs from HIV+ patients while TNFand IL1 were not detected suggesting an immunomodulatory effect may be elicited by the EVs. Other immune downregulating factors, IDO, HOXB4, HGF, and TGF 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 -1, an inhibitor of immune function, is induced by HIV Tat [61] and is a mediator of immune suppression in HIV infection [62][63][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 -1 and IL10 than patients with progressive disease [65] and it is possible that EVs may sequester TGF -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 naïve 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.

Conflicts of Interest
The authors declare that they have no conflicts of interest.