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

Detection of HIV-1 and Human Proteins in Urinary Extracellular Vesicles from HIV Hindawi Advances in Virology Volume 2018, Article ID 7863412, 16 pages https://doi.org/10.1155/2018/7863412 Research Article Detection of HIV-1 and Human Proteins in Urinary Extracellular Vesicles from HIV+ Patients 1 1 1 Samuel I. Anyanwu, Akins Doherty, Michael D. Powell, 2 1 3 Chamberlain Obialo, Ming B. Huang, Alexander Quarshie , 1 2 1 Claudette Mitchell, Khalid Bashir, and Gale W. Newman Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, GA, USA Department of Medicine, Morehouse School of Medicine, Atlanta, GA, USA 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 Copyright © 2018 Samuel I. Anyanwu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Extracellular vesicles (EVs) are membrane bound, secreted by cells, and detected in bodily uids, fl 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 )andHIV− (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. 1. Introduction only accounts for 0.01% of urine components; however, in certain disease states, the protein content and EV numbers Extracellular vesicles (EVs) are membrane bound vesicles, can increase in urine [12–16]. The glomerular capsule filters between 30 nm and 1𝜇minsize, aresecretedintoblood, bloodthatispassedintotherenaltubuleandaccounts for urine, saliva, semen, and other bodily uids, fl and have been thirty percent of the urinary protein content [14–16]. eTh suggested as a potential source of biomarkers for disease pro- remaining seventy percent of proteins in urine is derived from gression [1, 2]. These EVs, microparticles and/or exosomes, the kidney [17], and thus, urinary EVs are comprised of both aresecretedbycellsnormally orwhiletheyareundergoing renal and efferent components. stress or apoptosis [3] and contain proteins, mRNA, and HIV proteins are detected in EVs of HIV+ patients and miRNA [4] that are involved in cell to cell communication, HIV Nef is the most prevalent protein found [18–21]. Other transfer of antigens to cells, and intracellular communication. reports of HIV proteins in EVs are from in vitro transfected EVs are described in cancer disease pathogenesis [5] in HIV or HIV infected cultured cells and are not from HIV+ patient infection [6], other viral infections [7], and other disease samples [6, 18, 19, 22, 23]. states such as cardiovascular, renal, liver, and metabolic Biomarkers in urinary EVs are suggested for use in the disease [8–11]. diagnosis of many disease states [12, 13, 24–30]. The objectives EVsfromurine areanattractivenoninvasive sourcefor of this study were to determine the differences in proteins biomarkers of diseases [12, 13]. In healthy individuals, protein from urinary EVs from HIV+ patients and HIV− individuals 2 Advances in Virology Table 1: Patient demographics. a JEOL 1200EX transmission electron microscope (JEOL, Peabody, MA). HIV-positive HIV-negative Characteristics (𝑁 =35) (𝑁 =12) .. Nanoparticle Tracking Analysis (NTA). Urine samples Age (median± IQR) 41.5± 14.25 59± 18 from HIV-negative (𝑛=8 )andpositive individuals(𝑛= Sex (𝑛,%) 11), 15 ml, were centrifuged at 300×gfor 10minat4 C Male 25 (71.4%) 7 (58.3%) to remove cell debris. eTh supernatant was collected and Female 10 (28.6%) 5 (41.7%) centrifuged at 16,500×gfor20minat4 Candthesuper- Race (𝑛,%) natant collected and ultracentrifuged at 120,000×gat4 C for 1.5 hr. eTh pellet was resuspended in 500 𝜇lofPBS. African American/Black 28 (80%) 12 (100%) eTh size and quanticfi ation of the EVs were analyzed using White 7 (20%) - the NanoSight NS500 (NanoSight NTA 2.3 Nanoparticle Hispanic - - Tracking and Analysis Release version build 0025). Particles Asian - - were automaticallytracked andsized basedonBrownian Viral loads (copies/ml) (median motion and the diffusion coefficient. The NTA measurement 50±0- ± IQR) ∘ conditions were temperature 21.0 +/− 0.5 C, viscosity 0.99 CD4+ T cell (cells/𝜇l) (median ± +/− 0.01 cP, frames per second 24.99–25, and measurement 66.5± 46.5 - IQR) time 30 s. The detection threshold was similar in all samples. Antiretroviral therapy (𝑛, %) 34 (97.1%) - Two recordings were performed for each sample. .. Mass Spectrometry Analysis. Thirty-five (35) HIV+ and twelve (12) HIV− EV samples were lysed and trypsinized using proteomics and mass spectrometry. eTh analysis of and the sequence of peptides was determined by tandem more patient samples could identify specific EV urinary proteins as biomarkers of HIV infection, treatment ecffi acy, mass spectrometry (LC/MS/MS), using an LTQ Ion Trap Mass Spectrometer (Thermo Fischer Scientific, Waltham, and/or disease progression. MA). Peptides were rfi st reduced in DTT 10 mM at 56 C foratleast 30minand alkylatedwith15mMiodoaceticacid 2. Methods for 30 min at room temperature in the dark. Samples were then digested with mass spectrometry grade trypsin 20 ng/𝜇l .. Sample Collection. Urine was collected from thirty-five for 4 hours at 37 C. Just before analysis, the sample was (35) HIV+ patients and twelve (12) HIV− individuals in sterile acidified by the addition of formic acid to 0.1%. Peptides collection cups. eTh subjects were recruited from clinics in were separated by reverse phase HPLC (Agilent) on a 0.5× the metropolitan Atlanta area, GA. Patient demographics are 75 mm C-18 column (Michrom) at a flow rate of 500 nl/min described in Table 1. eTh study was approved by the Institu- using a linear gradient of acetonitrile (5–35%) over 100 min. tional Review Board of Morehouse School of Medicine and Ions were directly introduced by nanospray and spectra written informed consent was obtained from all participants. were collected using Xcalibur 2.0 software using an intensity thresholdof200counts.Theresulting spectrawereanalyzed .. EV Isolation. Urine samples were centrifuged at 1000×g using Bioworks 1.1 software to search a hybrid Human-HIV to remove cells and sediment then frozen at−80 C. Samples, database created from the complete nonredundant peptide 4 ml, were thawed and the EVs isolated followed by centrifu- database from NCBI. eTh threshold for inclusion in the gal filtration using Amicon Ultra-4 100 kDa centrifugal filter search is a minimal S/N ratio of 3. False discovery rates were unit (Millipore, Billerica, MA), at 3000×gfor15minutesat determined and set based on the control HIV− samples. An 4 C. The retentate, containing EVs, was collected from the initial protein identification list was generated from matches top of the filter and resuspended in 200 𝜇lphosphate bueff red with an Xcorr score versus charge state of 1.0 (+1) 1.5 (+2) and saline (PBS) for use in the transmission electron microscopy 1.7 (+3) and consensus scores greater than 10.0. and tandem mass spectrometry (LC/MS/MS) analysis. Bioinformatics techniques for analysis of HIV EV pro- teins were used on the LC/MS/MS detected proteins [31]. .. Transmission Electron Microscopy Analysis. Transmis- Functional enrichment analysis was performed using Fun- sion electron microscopy (TEM) was used to identify EVs in Rich (Functional Enrichment analysis tool, http://funrich two HIV-1 positive and two HIV-1 negative samples. Urinary .org/index.html) [32] against a human database to detect pro- EVswerefixedin2.5%glutaraldehydein0.1Mcacodylate teins involved in biological processes, cellular components, buffer for 2 hours at 4 Cfollowedby2washeswith 0.1M sites of expression, and biological pathways. Only processes cacodylate buffer, 5 minutes each. Samples were stained with with a𝑃 value< 0.05, using the Benjamini-Hochberg False 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 hour at Discovery rate, were reported. eTh human proteins detected 4 C followed by 2 washes with the cacodylate bueff r and 3 were compared to the top 100 EV proteins in ExoCarta washes with deionized water, 5 minutes each. Samples were (http://exocarta.org/exosome markers new) [33, 34], sixty subsequently stained with 0.5% aqueous uranyl acetate for EV proteins in the EV array [35], and proteins identified in 2 hours at room temperature and subsequently viewed with EVs from HIV infected lymphocytic cells [36]. Advances in Virology 3 Pathway analysis comparing HIV+ samples with CD4+ Rev, reverse transcriptase, Tat, Vif, Vpr, and Vpu. All HIV+ T cells greater than 300 (𝑛=15 ) to those with less than urine samples (𝑛=35 ) contained at least one HIV-1 protein in 500 (𝑛=15 )andHIVhighVL,greaterthan200 copies EVs, while no HIV proteins were found in the HIV− samples (𝑛=10 ), compared to HIV low viral loads, less than 200 (𝑛=12 ) (Table 3). HIV-1 Nef was detected in twenty-six copies (𝑛=10 ), was done using Pathway Studio version of thirty-five (26 of 35) (74.3%) HIV+ urine samples. Three 11.4 Mammal Plus (Elsevier, Inc., Atlanta, GA). Gene Set (3) patients’ urine samples, #173, #174, and #196, were tested Enrichment Analysis (GSEA) was used to identify the top 10 203, 311, and 35 days, respectively, aer ft their first EV sample curated pathways for the proteins in the each of the patient was analyzed. No difference in the HIV proteins detected in groups. No comparisons were done between patients not on sample #196, 35 days aeft r his previous sample, was found. ART or undergoing ART because there was only one patient #173’s sample,tested203 days aeft r thefirstanalysis, hada not on ART. similar profile, except that Rev and Tat were not detected. In e Th HIV proteins, Nef, Vpr, Vpu, and Vif, were searched addition, #174’s EVs examined 311 days aer ft the first sampling using the HIV-1 Human Interaction database (https://www foundRev andRTmissingfromtheprofile. .ncbi.nlm.nih.gov/genome/viruses/retroviruses/hiv-1/inter- HIV p24 antigen was only detected in ve fi of thirty-ve fi actions/). This database contains all the known, published (5 of the 35 patient) (14%) samples by LC/MS/MS, but of the interactions of HIV-1 gene products with human proteins twenty-six (26) HIV+ and eleven (11) HIV-negative samples [37]. Proteins from the search were compared to the human tested by ELISA, no p24 was detected. There was no statistical proteins detected in the HIV EVs. correlation of the number of HIV proteins detected with CD4+ T cell counts, viral loads, or ART therapy. .. Western Blot Analysis. To validate the presence of HIV Validation by WB analysis using polyclonal pooled proteins in urinary EVs, western blot analysis (WB) was patient serum and monoclonal antibodies against HIV Nef performedontwenty(20)randomlyselectedHIV+and andHIV p24indicatedthepresenceofHIV proteins.Figure1 three (3) HIV− control urine samples. Recombinant HIV-1 is a WB using polyclonal pooled HIV+ serum used as the Nef and HIV-1 p24 were used as positive controls, while detection antibody. All the HIV+ patient samples contained HIV-negative urine and HIV-positive filtrate were used HIV-1 proteins and the top panel shows patient samples as negative controls. Samples were heated at 85 Cfor two reacting to anti-HIV Nef. HIV+ urine samples, 7 of 9 (77.7%), minutes in a tris-glycine SDS sample buffer, were loaded into showed HIV-1 Nef bands at 27 kD. a 4–20% TGX gradient gel (Bio-Rad, Hercules, CA), and run for 50 mins at 200 V. A semidry transfer unit (Hoefer .. TEM and NTA Analysis of EVs. TEManalysisofurine Scientific, Holliston, MA) was used to transfer the separated from HIV+ patients showed multiple EVs, ranging in size proteins onto a PVDF membrane (Bio-Rad) at 15 V for from 50 nm to 300 nm (Figure 2(a)), while two HIV-negative 50 mins. eTh filter was blocked for nonspecific binding controls had fewer EVs present (Figure 2(b)). NTA analysis using 5% nonfat dry milk in 1x tris buffered saline (TBS) showed that there were signicfi antly more EVs from HIV+ with Tween 20. The membrane was incubated overnight in patients than healthy controls,4.96±0.0733 and3.69±0.075, pooled plasma from twenty HIV+ patients as the primary respectively (𝑃<0.05). No significant differences were found antibody at a 1 : 500 dilution and rabbit anti-human IgG in the size of the EVs, 110–227 nm for HIV-negative donors conjugated HRP antibody (1 : 1000, Bio-Rad, Hercules, CA) and 54–448 nm HIV+ samples. Representative Nanosight wasusedassecondaryantibody.Super Signal West Femto analyses for HIV-negative and HIV+ urine samples are (Thermo Fischer Scientific, Waltham, MA) was used as a showninFigure3. chemiluminescent substrate for detection. eTh membrane wasdeveloped andimagedusingtheLAS 4000biomolecular .. Human Proteins in HIV+ and Negative EV Urine Sam- imager (GE Healthcare Life Sciences, Pittsburgh, PA). ples. EV proteins from the HIV+ patients, 14,475, which Recombinant HIV-1 Nef and p24 WB analyses were detected enteredintoFunRich,functionalenrichmentanalysisso-ft using anti-Nef and p24 monoclonal antibodies (1 : 500, EMD ware, showed 29.44% or 1,932 proteins were associated with Millipore, Billerica, MA) and anti-mouse IgG conjugated exosomes (Table 4). es Th e EV identified proteins were com- HRP antibodies (1 : 1000, Bio-Rad, Hercules, CA) were used. pared to top 100 EV proteins in the ExoCarta database with 83% matching (http://exocarta.org/exosome markers new) .. HIV p ELISA. Twenty-six (26) HIV+ and eleven (11) [33], 22 EV proteins in the EV array [35] were similar, and HIV− urine samples were tested for the presence of HIV p24 7 of 14 EV proteins identified in exosomes from HIV infected by ELISA (ImmunoDX, Woburn, MA). lymphocytes [36] were found and are highlighted in Table 4. Exosomal proteins found in the control samples are listed in Table 5. 3. Results eG Th O resultsoftheFunRichanalysisoftheEVs .. HIV Proteins Are Present in Urinary EVs of HIV-Positive from the HIV+ samples are summarized in Table 6 and Patients. LC/MS/MS mass spectrometry HIV EV protein Figure 4. The top vfi e ( 𝑃 < 0.01) EV sites of expression results are presented in Table 2. Urinary EV proteins meeting were endothelial cells, plasma, liver, serum, and kidney and the false discovery rate and Xcorr score criteria as HIV-1 themostsignicfi antcellularcomponentswerelysosomes, proteins included Nef, Gag, Pol, Protease, gp120, gp160, gp41, exosomes, membranes, plasma membranes, the nucleus, and 4 Advances in Virology Table 2: LC/MS/MS analysis of EV HIV proteins. Accession # AAs MW [kDa] Calc. pI Description Σ Coverage Σ# peptides Score A0 Coverage A0 # peptides A0 gi38491705 192 22.7 10.1 Vif protein [human immunodeficiency virus 1] 13.54 12 9.22 13.54 4 gi73913089 104 11.7 10.1 Gag protein [human immunodeficiency virus 1] 14.42 15 6.27 14.42 4 gi58374258 869 98.1 8.8 Envelope glycoprotein [human immunodeficiency virus 1] 1.5 5 5.63 1.5 3 gi183197180 404 45.8 8.4 Pol protein [human immunodeficiency virus 1] 3.47 3 5.27 3.47 3 gi255984636 160 18.1 5.3 Reverse transcriptase [human immunodeficiency virus 1] 7.5 4 4.80 7.5 2 gi256012108 114 13.5 5.7 Nef protein [human immunodeficiency virus 1] 14.04 3 4.53 14.04 2 gi9756252 524 60.4 8.7 Pol precursor [human immunodeficiency virus 1] 4.01 2 4.46 4.01 2 gi67082579 191 22.3 9.4 Reverse transcriptase [human immunodeficiency virus 1] 10.47 2 4.43 10.47 2 gi2290009 852 96.7 8.5 Envelope glycoprotein [human immunodeficiency virus 1] 7.16 11 4.33 5.87 3 gi167886806 25 2.7 8.7 Rev protein [human immunodeficiency virus 1] 56 4 4.29 56 2 gi23344577 99 10.6 9.4 Protease [human immunodeficiency virus 1] 12.12 5 4.36 12.12 2 gi4324808 1437 161.9 8.3 Gag-pol polyprotein [human immunodeficiency virus 1] 2.51 7 4.05 1.6 2 gi222533599 73 8.0 9.1 Env C2V3 protein [human immunodeficiency virus 1] 23.29 4 3.85 23.29 2 gi71060450 206 23.7 6.3 Negative factor [human immunodeficiency virus 1] 4.85 4 3.84 4.85 2 gi37935985 85 10.3 4.8 Vpu protein [human immunodeficiency virus 1] 11.76 4 3.84 11.76 2 gi108860432 870 98.8 8.5 gp160 [human immunodeficiency virus 1] 3.33 2 3.74 3.33 2 gi114801226 209 24.4 10.1 Tat protein [human immunodeficiency virus 1] 5.26 2 3.70 5.26 2 gi22596451 341 38.6 8.0 Truncated envelope glycoprotein [human immunodeficiency virus 1] 3.81 4 3.68 3.81 2 gi183200570 342 38.7 9.2 Truncated pol protein [human immunodeficiency virus 1] 3.51 2 3.68 3.51 2 gi34786230 176 19.8 9.6 gp120 protein [human immunodeficiency virus 1] 10.8 3 3.60 10.8 3 gi1002239 104 11.5 8.9 Envelope glycoprotein, v3 region [human immunodeficiency virus 1] 18.27 2 3.54 18.27 2 gi77168129 95 11.3 7.6 Vpr protein [human immunodeficiency virus 1] 13.68 3 3.40 13.68 2 gi222532593 129 14.7 8.4 Gag p17 protein [human immunodeficiency virus 1] 8.53 3 2.61 8.53 1 gi219688191 132 14.9 10.2 Matrix protein [human immunodeficiency virus 1] 9.09 6 2.56 9.09 1 gi255687141 288 32.1 7.7 Integrase [human immunodeficiency virus 1] 9.03 1 2.52 9.03 1 gi222532161 132 15.0 9.5 Gag p17 protein [human immunodeficiency virus 1] 9.09 6 2.45 9.09 1 gi37934078 573 65.0 9.0 Gag-pol fusion polyprotein [human immunodeficiency virus 1] 2.79 2 2.42 2.79 2 gi405003 207 23.1 7.7 gp120 [human immunodeficiency virus 1] 12.08 1 2.41 12.08 1 gi54792352 213 23.7 5.6 Gag polyprotein [human immunodeficiency virus 1] 9.39 2 2.34 9.39 2 gi3885826 132 14.9 9.6 p17 matrix [human immunodeficiency virus 1] 11.36 2 2.31 11.36 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. Advances in Virology 5 Table 3: Presence of HIV-1 proteins in HIV+ patient urinary EVs. Viral load CD4 ID ART AIDS Nef Gag Pol Protease Rev RT Tat Vif p1 p24 p17 Poly Vpu Env Vpr Vif cells/ul copies/ml 22 No XX X X X X X X X X 27 Yes AIDS <50 X 28 Yes AIDS 280100 XX X X X 30 Yes AIDS >10000 XX X X X X X X XX <20 41 Yes 29187 XX X 46 Yes <50 XX X X 45 Yes 400 XX X X X 48 Yes 4974 XX XX 51 NA XX X X X NA 52 Yes 51 XX X X X X X X 61 <50 XX 62 Yes AIDS <50 X 63 No 2023 X 65 No NA X NA 66 No NA X NA 67 Yes 75 X 68 No NA X NA 69 Yes <50 X 70 Yes <50 X 71 Yes <50 456 X 74 NA X NA 86 Yes <75 XX X X 103 Yes AIDS 150 XX X 104 Yes AIDS 77 313 XX 108 Yes AIDS <50 XX X 110 Yes <50 XX 111 Yes AIDS <50 XX 112 Yes AIDS >200 581 XX 142 Yes <50 XX X X X X X X 173-1 Yes <50 XX X X X X X X X X 173-2 Yes <50 XX X X X X 174-1 Yes 48 315 XX X X X X 174-2 Yes 48 XX X X 196-1 Yes AIDS <50 XX X X X X X X X X X X X 196-2 Yes AIDS <50 XX X X X X X X X X X 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 consens us scores greater than 10.0; NA = not available. the cytoplasm(𝑃<0.01) (Figure 4). The top vfi e ontologies T cells less than 300, 15,028 proteins from patients with low (Table 6) were protein serine/threonine kinase activity, cat- VL, and 2486 from patients with high VLs. Pathway analysis alytic activity, GTPase activator activity, guanyl-nucleoside was similar between EV proteins from patients with greater exchange factor activity, and cell adhesion molecule activity than 300 CD4+ T cells and low VLs and different between (𝑃 < 0.0001), the top biological process was regulation of the low CD4+ T cells and high VLs (summarized in Table 7). nucleobase, nucleoside, and nucleic acid (𝑃< 0,0001), and the The pathways found are detailed in Supplementary Material most prominent biological pathway was integrin cell surface 1. Interleukin proteins detected were IL10, IL10RA, IL16, interactions (𝑃<0.03). IL17RC, IL18, IL18BP, IL1RAP, IL1RL2, IL1RN, IL33, IL4I1, LC/MS/MS identified 15,571 proteins in EVs from HIV+ IL6, and IL6ST. Immunomodulatory molecules, HOXB4, patients with CD4+ T cells greater than 300, 2,115 from CD4+ CD81,CD9,TGF-𝛽1, IDO, Notch1, ADAM17, Rab4, and HGF, 6 Advances in Virology Table 4: Exosomal proteins found in urinary EVs from HIV+ patients. Genes in our analysis Genes in the FunRich database Percentage of genes Fold enrichment Exosomal proteins 1932 2001 29.44 2.11 A1BG, AM, AARS, ABCA7, ABCB1, ABCB11, ABCB6, ABCC1, ABCC11, ABCC9, ABCG2, ABHD8, ACAA2, ACAT1, ACAT2, ACE, ACE2, ACLY, ACO1, ACOT11, ACP2, ACSL3, ACSL4, ACSM1, ACTA1, ACTA2, ACTB, ACTBL2, ACTC1, ACTG1, ACTL6A, ACTN1, ACTN2, ACTN4, ACTR1A, ACTR1B, ACTR2, ACTR3, ACY1, ACY3, ADAM10, ADAMTS3, ADCY1, ADH5, ADH6, ADK, ADSL, AEBP1, AGAP2, AGR2, AGR3, AGRN, AGT, AHCTF1, AHCYL1, AHNAK, AHSA1, AHSG, AK1, AK2, AKAP9, AKR1A1, AKR1B10, ALAD, ALB, ALCAM, ALDH16A1, ALDH1A1, ALDH1L1, ALDH2, ALDH3B1, ALDH8A1, ALDOA, ALDOB, ALDOC, ALK, ALOX12, ALPL, ALPP, ALYREF, AMBP, AMN, ANGPT1, ANGPTL1, ANGPTL4, ANKFY1, ANKRD11, ANO1, ANO6, ANPEP, ANXA, ANXA, ANXA13, ANXA3, ANXA, ANXA, ANXA7, AOX1, AP1M1, AP2A1, AP2A2, AP2M1, AP4M1, APAF1, APLP2, APOA1, APOA2, APOB, APOD, APOE, APOL1, APP, APPL1, APPL2, APRT, AQP2, ARF5, ARFIP1, ARHGAP1, ARHGAP23, ARHGDIA, ARHGDIB, ARHGEF12, ARHGEF18, ARL15, ARL3, ARL8B, ARMC3, ARMC9, ARPC1A, ARPC1B, ARPC2, ARPC3, ARPC5, ARRDC1,ARSE,ARSF,ARVCF,ASAH1,ASB6, ASL,ASNA1, ASNS,ATAD2,ATIC, ATPA, ATP1A2, ATP1A3, ATP2B1, ATP2B2, ATP2B4, ATP4A, ATP5A1, ATP5B, ATP5L, ATP6AP1, ATP6AP2, ATP6V0A1, ATP6V0A4, ATP6V0C, ATP6V0D1, ATP6V0D2, ATP6V1A, ATP6V1B1, ATP6V1C1, ATP6V1C2, ATP6V1D, ATP6V1E1, ATP6V1H, ATRN, AUP1, AZGP1, AZU1, B2M, B3GAT3, B4GALT1, B4GALT3, BAIAP2, BAIAP2L1, BASP1, BAZ1B, BCAM, BCR, BDH2, BGN, BHLHB9, BHMT, BHMT2, BLMH, BLOC1S5, BLVRA, BLVRB, BMP3, BPI, BPIFB1, BPTF, BRI3BP, BROX, BSG,BTG2, BTN1A1,C11orf52,C11orf54,C16orf80,C16orf89,C17orf80, C19orf18,C1GALT1C1, C1orf116, C1QC, C1QTNF1, C1QTNF3, C1R, C2orf16, C3, C4BPA, C5, C9, CAB39L, CACNA2D1, CACYBP, CAD, CALM1, CALML3, CALR, CAMK4, CAMP, CAND1, CANX, CAP1, CAPN1, CAPN2, CAPN5, CAPN7, CAPNS1, CAPS, CAPZA2, CAPZB, CARD11, CASP9, CAV1, CBR3, CC2D1A, CCDC105, CCDC132, CCL28, CCPG1, CCT, CCT, CCT4, CCT, CCT6A, CCT7, CCT8, CD101, CD14, CD163L1, CD19, CD2, CD22, CD274, CD2AP, CD300A, CD36, CD37, CD40, CD44, CD53, CD55, CD58, CD59, CD63, CD70, CD74, CD79B, CD80, CD81, CD9,CD97, CDC, CDC42BPA, CDC42BPB, CDH1, CDH17, CDHR2, CDHR5, CDK1, CDK5RAP2, CDKL1, CEACAM5, CELSR2, CEMIP, CEP250, CES2, CETP, CFD, CFH, CFI, CFL, CHGB, CHID1, CHMP1A, CHMP2B, CHMP4B, CHRDL2, CHST1, CHST14, CIB1, CKAP4, CKB, CLASP1, CLCA4, CLDN3, CLDN4, CLDN7, CLIC1, CLIC4, CLIC5, CLIC6, CLIP2, CLSTN1, CLTC, CLTCL1, CLU, CMPK1, CNDP2, CNKSR2, CNTLN, COASY, COBLL1, COL12A1, COL15A1, COL18A1, COL6A1, COL6A2, COL6A3, COLEC10, COLGALT1, COMT, COPA, COPB1, COPB2, COPS8, CORO1A, CORO1B, COX4I1, COX5B, CP, CPD, CPN2, CPNE1, CPNE3, CPNE5, CPNE8, CPVL, CR1, CR2, CRB2, CREB5, CRISPLD1, CRNN, CRTC2, CRYAB, CRYZ, CS, CSE1L, CSK, CSPG4, CSRP1, CST4, CSTB, CTDSPL, CTNNA1, CTNNB1, CTNND1, CTSB, 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, DDX11, DDX19A, 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, DNAJC7, DNHD1, DNM2, DNPH1, DOCK10, DOCK2, DOPEY2, DPEP1, DPP3, DPP4, DPYS, DPYSL2, DSC2, DSG2, DSG3, DSP, DSTN, DUOX2, DUSP26, DUT, DYNC1H1, DYNC2H1, DYSF, ECE1, ECH1, ECM1, EDIL3, EEA1, EEFA, EEF1A2, EEF1D, EEF1G, EEF, EFEMP1, EFEMP2, EGF, EGFR, EHD1, EHD2, EHD3, EHD, EIF2S1, EIF2S3, EIF3A, EIF3B, EIF3E, EIF3L, EIF4A1, EIF4A2, EIF4A3, EIF4E, EIF4G1, EIF4H, ELANE, EML5, ENO, ENO2, ENO3, ENPP3, ENPP4, ENPP6, ENTPD1, EPB41L2, EPCAM, EPHA2, EPHA5, EPHB1, EPHB2, EPHB3, EPHB4, EPHX2, EPN3, EPPK1, EPRS, EPS8, EPS8L1, EPS8L2, EPS8L3, ERAP1, ERBB2, ERMN, ERO1L, ERP44, ESD, ETFA, EVPL, EXOC4, EXOSC10, EXT2, EYS, EZR, F11, F11R, F5, F7, FABP1, FABP3, FAH, FAM129A, FAM129B, FAM151A, FAM208B, FAM209A, FAM20A, FAM20C, FAM49B, FAM65A, FAS, FASLG, FASN, FAT1, FAT2, FBL, FBP1, FBP2, FCGBP, FCN1, FCN2, FERMT3, 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, G6PD, GAA, GABRB2, GAL3ST4, GALK1, GALM, GALNT3, GANAB, GARS, GART, GATSL3, GBE1, GBP6, GCN1L1, GCNT2, GCNT3, GDF2, GDI, GDPD3, GEMIN4, GFPT1, GGCT, GGH, GGT1, GHITM, GIPC1, GK, GK2, GLB1, GLDC, GLG1, GLIPR2, GLO1, GLUD1, GLUL, GNA13, GNAI1, GNAI,GNAQ, GNAS, GNB, GNB, GNB2L1, GNB3, GNB4, GNB5, GNG12, GNPDA1, GNPTG, GOLGA4, GOLGA7, GOT1, GOT2, GPC1, GPC4, GPD1, GPI, GPM6A, GPR155, GPR64, GPR98, GPRASP1, GPRC5A, GPRC5B, GPT, GREB1, GRHPR, GRID1, GRIN1, GRK4, GSN, GSR, GSS, GSTA3, GSTCD, GSTK1, GSTO1, GSTP1, GUSB, H1FOO, H2AFY, H2AFY2, HADHA, HAPLN3,HAUS5,HBB,HBD,HBS1L,HDHD2, 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, HSP90AB1, HSP90B1, HSPA12A, HSPA13, HSPA1L, HSPA2, HSPA4, HSPA,HSPA6, HSPA , HSPA9, HSPB1, HSPB8, HSPD1, 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, IST1, ITFG3, ITGA1, ITGA2, ITGA2B, ITGA3, ITGA4, ITGA,ITGAL,ITGAV, ITGB,ITGB2,ITGB3,ITGB4,ITGB7,ITGB8,ITIH2,ITIH4,ITM2C, ITSN1,ITSN2,IVL, JADE2,JUP, KALRN, KCNG2,KHK, KIAA1324, KIF12, KIF15,KIF18B,KIF3A, KIF3B, KIF9,KIFC3,KL, KNG1, KPNB, KPRP, KRT1, KRT10, KRT12, KRT14, KRT15, KRT16, KRT17, KRT18, KRT19, KRT2, KRT20, KRT24, KRT25, KRT27, KRT28, KRT3, KRT5, KRT6C, KRT7, KRT73, KRT75, KRT76, KRT77, KRT78, KRT79, KRT8, KRT9, L1CAM, LAD1, LAMA3, LAMA4, LAMA5, LAMB2, LAMB3, LAMC1, LAMC2, LAMP1, LAMP2, LAMTOR3, LBP, LCK, LCP1, LDHA,LDHB,LEPRE1, LFNG,LGALS3, LGALSBP, LGALS4, LIMA1, LIN7A, LIN7C, LMAN1, LMAN2, LOXL4, LPO, LRP1, LRP1B, LRP2, LRP4, LRPPRC, LRRC15, LRRC16A, LRRC57, LRRK2, LRSAM1, LSP1, LSR, LTA4H, LTBP3, LTF, LUZP1, LYPLA2, MAGI3, MAL2, MAN1A1, MAN1A2, MAN2A1, MAP4K4, MAP7, MARCKSL1, MARK3, MARS, MARVELD2, MASP1, MASP2, MBD5, MBLAC2, MCAM, MCPH1, MDH1, MDH2, MEGF8, MEP1A, MEST, METRNL, MFGE , MFI2, MGAM, MGAT1, MGAT4A, MID2, MIF, MINK1, MLLT3, MLLT4, MME, MMP24, MMP25, MMRN1, MMRN2, MNDA, MOB1A, MOB1B, MOGS, MPO, MPP5, MPP6, MS4A1, MSH6, MSN, MSRA, MTA1, MTAP, MTCH2, MTHFD1, MTMR11, MTMR2, MUC13, MUC16, MUC4, MUM1L1, MVB12A, MVB12B, MVP, MX1, MXRA5, MXRA8, MYADM, MYH10, MYH11, MYH13, MYH14, MYH3, MYH8, MYH , MYL6B, MYO15A, MYO1B, MYO1C, MYO1D, MYO1E, MYO1G, MYO5B, MYO6, MYOF, N4BP2L2, NAA16, NAA50, NACA, NAGLU, NAMPT, NAP1L4, NAPA, NAPG, NAPRT, NAPSA, NARS, NBR1, NCALD, NCCRP1, NCKAP1, NCKAP1L, NCL, NCOA3, NCSTN, NDRG1, NDRG2, NEB, NEBL, NEDD4, NEDD4L, NEDD8, NEU1, NID1, NIN, NIPBL, NIT2, NKX61, NONO, NOTCH1, NOX3, NPC1, NPEPPS, NPHS1, NPHS2, NPM1, NPNT, NQO2, NT5C, NT5E, NUCB1, NUCB2, NUDT5, NUMA1, NXPE4, OLA1, OPTN, OR2A4, OS9, OSBPL1A, OXSR1, P2RX4, P4HB, PA2G4, PACSIN2, PACSIN3, PADI2, PAFAH1B1, PAFAH1B2, PAGE2, PAICS, PAM, PARD6B, PARP4, PBLD, PCBP1, PCDHGB5, PCK1, PCLO, PCNA, PCSK9, PCYOX1, PDCD2, PDCD5, PDCD6, PDCD6IP, PDDC1, PDE8A, PDIA2, PDIA3, PDIA4, PDIA6, PDLIM7, PDZK1, PEBP1, PECAM1, PEF1, PEPD, PEX1, PFAS, PFKL, PFKP, PGAM1, PGD, PGK, PGLYRP1, PGM1, PHB2, PHGDH, PI4KA, PIGR, PIK3C2A, PIK3C2B, PILRA, PIP, PIP4K2C, PKD1, PKD1L3, PKD2, PKHD1, 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, PMEL, PNP, PODXL, POFUT2, PON1, PON3, POTEE, POTEF, POTEI, POTEM, PPA1, PPARG, PPFIA2, PPIA, PPIB, PPL, PPM1L, PPP1CB, PPP1R7, PPP2CA, PPP2R1A, PPP2R1B, PRCP, PRDX, PRDX3, PRDX4, PRDX5, PRG4, PRKAR2A, PRKCA, PRKCD, PRKCH, PRKCI, PRKCZ, PRKDC, PRKRIP1, PRNP, PROM1, PROM2, PROS1, PROZ, PRRC2A, PRSS23, PRTN3, PSAP, PSAT1, PSMA2, PSMA3, PSMA5, PSMA7, PSMB1, PSMB3, PSMB4, PSMB5, PSMB6, PSMB8, PSMB9, PSMC2, 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,QDPR,QPCT,QPRT, QSOX1, RAB10, RAB11B, RAB17, RABA, RAB1B, RAB22A, RAB25, RAB29, RAB2A, RAB34, RAB3B, RAB3GAP1, RAB43, RAB4B, RAB6B, RABA, RAB A,RAB8B,RAB9A, RAC, RACGAP1, RALA, RALB, RAP1A, RAPB, RAP1GDS1, RAP2A, RAPGEF3, RARRES1, RARS, RASAL3, RASSF9, RBL2, RCC2, REG4, RELN, RENBP, RFC1, RFTN1, RHEB, RHOB, RHOF, RIMS2, RLF, RNASE7, RNF213, RNH1, RNPEP,ROBO2,ROCK2,RP2,RPL10,RPL10A,RPL14,RPL15, 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, RRAS, RREB1, RSU1, RTN4, RUSC2, RUVBL1, RUVBL2, RYR1, S100A11, S100A6, S100P, SAA1, SAFB2, SAMM50, SARS, SBSN, SCAMP2, SCAMP3, SCARB1, SCARB2, SCEL, SCIN, SCN10A, SCN11A, SCPEP1, SCRIB, SCRN2, SDCBP, SDF4, SEC31A, SELENBP1, SELP, SEMA3G, SEPP1, SERBP1, SERINC1, SERINC2, SERINC5, SERPINA1, SERPINA3, SERPINA4, SERPINA5, SERPINA7, SERPINB1, SERPINB13, SERPINB6, SERPINB9, SERPING1, SETD4, SFI1, SFN, SFRP1, SFT2D2, SH3BP4, SHMT1, SHMT2, SHROOM2, SIAE, SIRPA, SIT1, SLAMF1, SLAMF6, SLC12A1, SLC12A2, SLC12A3, SLC12A7, SLC12A9, SLC13A2, SLC13A3, SLC15A2, SLCA, SLC1A1, SLC1A4, SLC1A5, SLC20A2, SLC22A11, SLC22A12, SLC22A13, SLC22A2, SLC22A5, SLC22A6, SLC23A1, SLC25A1, SLC25A3, SLC25A4, SLC25A6, SLC26A11, SLC26A4, SLC26A9, SLC27A2, SLC29A1, SLC2A1, SLC2A3, SLC34A2, SLC35D1, SLC36A2, SLC37A2, SLC38A1, SLC39A5, SLC3A1, SLCA,SLC44A1, SLC44A2, SLC44A4, SLC46A3, SLC4A1, SLC4A4, SLC5A1, SLC5A10, SLC5A2, SLC5A5, SLC5A6, SLC5A8, SLC5A9, SLC6A13, SLC6A14, SLC6A19, SLC7A5, SLC9A1, SLC9A3, SLC9A3R1, SLC9A3R2, SLCO4C1, SLIT2, SLK, SMC2, SMC3, SMIM22, SMIM24, SMO, SMPDL3B, SMURF1, SNCG, SND1, SNRNP200, SNX12, SNX18, SNX25, SNX33, SNX9, SOD1, SOGA1, SORD, SORL1, SORT1, SPAG9, SPAST, SPEN, SPINK1, SPON2, SPRR3, SPTAN1, SPTBN1, SQSTM1, SRC, SRPR, SRSF7, ST13, ST3GAL1, ST3GAL6, STAMBP, STAU1, STIP1, STK10, STK11, STK24, STOM, STRIP1, STX3, STX4, STX7, STXBP1, STXBP2, STXBP3, STXBP4, SUB1, SUCLA2, SUSD2, SYAP1, SYNE1, SYNE2, TAB3, TACSTD2, TAF6L, TALDO1, TAOK1, TARS, TAX1BP1, TAX1BP3, TBC1D10A, TBC1D21, TC2N, TCP, TECTA, TEKT3, TEX14, TF, TFRC, TGFB1, TGFBI, TGFBR3, TGM1, TGM2, TGM3, TGM4, THBS, THBS2, THRAP3, THSD4, THY1, TIAM2, TINAGL1, TJP2, TKT, TLN1, TLR2, TM7SF3, TM9SF2, TMBIM1, TMC6, TMC8, TMED2, TMED9, TMEM109, TMEM192, TMEM2, TMEM256, TMEM27, TMEM63A, TMPRSS11B, TMPRSS11D, TMPRSS2, TNFAIP3, TNFRSF8, TNFSF10, TNFSF13, TNIK, TNKS1BP1, TNPO3, TOLLIP, TOM1, TOM1L2, TOMM70A, TOR1A, TOR1B, TOR3A, TPI, TPM3, TPP1, TPRG1L, TRAP1, TREH, TRIP10, TSNAXIP1, TSPAN1, TSPAN15, TSPAN3, TSSK3, TSTA3, TTC17, TTC18, TTLL3, TTN, TTR, TUBAB, TUBA4A, TUBB3, TUBB4A, TUBB8, TUFM, TWF2, TXNDC16, TXNDC8, TXNRD1, TYK2, TYRP1, UACA, UBA, UBAC1, UBASH3A, UBE2N, UBE2V2, UBL3, UBXN6, UEVLD, UGDH, UGGT1, UGP2, ULK3, UMOD, UPB1, UPK1A, UPK3A, UQCRC2, UTRN, UXS1, VAMP1, VAMP3, VAMP7, VAPA, VASN, VASP, VAT1, VCL, VCP, VDAC3, VIL1, VIM, VMO1, VPS13C, VPS13D, VPS28, VPS35, VPS36, VPS37B, VPS37C, VPS37D, VPS4A, VPS4B, VTA1, VWA2, VWF, WARS, WAS, WASF2, WASL, WDR1, WIZ, WNT5B, XDH, XPNPEP2, XPO1, 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]. Advances in Virology 7 훼-HIV Nef 250 kD 훼-HIV antigens Patient ID 27 28 30 41 108 104 103 86 48 Nef c1 c2 c3 p24 MW kD Figure 1: Detection of HIV- 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. (a) (b) 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.EVsfromHIV-1posi. Table 5: Exosomal proteins found in urinary EVs from uninfected controls. Genes in our Genes in the FunRich Fold enrichment Percentage of genes analysis database Exosomal proteins 37 2001 72.54 5.26 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. 8 Advances in Virology Table 6: Functional enrichment analysis of HIV+ EV proteins. Genes in the Genes in the Percentage of Corrected𝑃 Fold enrichment dataset Bkg. database genes value (BH FDR) Molecular function Protein serine/threonine kinase −08 272 5,602 30 1.18 1.04 activity −05 Catalytic activity 456 827 4.9 1.1 1.12 −05 GTPase activator activity 131 836 4.7 1.2 8.14 Guanyl-nucleoside exchange −05 105 614 3.6 1.2 8.54 factor activity Cell adhesion molecule activity 307 531 3.3 1.1 0.0001 Biological process Regulation of nucleobase, −05 2,236 4,658 24.8 1.05 3.24 nucleoside, and nucleic acid Biological pathway Integrin cell surface interactions 69 1,366 23.3 1.2 0.03 Table 7: Comparison of pathways between HIV+ groups from Pathway Studio 11.4. HIV group Pathway Natural killer cell inhibitor receptor signaling Intermediate filament polymerization Ca2+ u fl x regulation G1/S phase transition CD4+ T cells greater than 300 G2/M phase transition 𝑛 =15 S/G2 phase transition Protein folding Golgi to endosome transport Endosomal recycling Kinetochore assembly Neutrophil chemotaxis Vascular motility Platelet activation via GPCR signaling Insulin influence on protein synthesis mTOR signaling overview CD4+ T cells less than 300 EDNRA/B→ vascular motility 𝑛 =15 Proplatelet maturation Natural killer cell activation through ITAM-containing receptors Taste sensor receptors activates mTOR signaling Natural killer cell activation Intermediate filament polymerization Natural killer cell inhibitory receptor signaling golgi to endosome transport Ca+ u fl x regulation Low VLs HRH1/3→ synaptic transmission 𝑛 =14 Vascular motility Endosomal recycling G1/S phase transition Golgi transport G2/M phase transition Metaphase/anaphase phase transition S/G2 phase transition Spindle assembly High VLs Natural killer cell activation 𝑛 =10 Histone ubiquitylation Eosinophil survival by cytokine signaling Protein folding G2/M phase transition Advances in Virology 9 Max intensity (AU): 23 0 100 200 300 400 500 600 700 800 900 0 100 200 300 400 500 600 700 (nm) Particle size/Concentration Particle size/Relative intensity (a) Max intensity (AU): 22118 0 100 200 300 400 500 600 700 0 100 200 300 400 500 600 700 800 900 Particle size/concentration (nm) Particle size/relative intensity (b) Figure 3: Nanosight analysis (representative analysis). (a) NTA analysis of an HIV-negative urine sample had 0.4× 10 particles per ml (left panel) while (b) depicts an urine sample from a HIV+ patient that had 8.7× 10 particles per ml and has a greater relative intensity profile (right panel (a) and (b)) when compared to the HIV-negative sample. eTh Rank Sum 𝑇 test showed that HIV+ patient urine samples had more particles per ml than the negative control urine (𝑃<0.05). were also found by LC/MS/MS in addition to MHC Class I processes were immune response, signal transduction, cell and II antigens. communication, and antigen presentation (𝑃<0.0073). The HIV-1 Human Interaction database search found that Only sixty-four (64) proteins overlapped between the HIV Nef interacted with 559 EV proteins of 770 total human HIV+ and control EV samples and are listed in Table 8. proteins (72.6%); HIV Vpr interacted with 437 EV of 598 The top fourteen (14) GO ontologies for cellular components human (73.1%); HIV Vif interacted with 162 EV of 310 human include extracellular exosome, extracellular region, extracel- (52.2%); and HIV Vpu interacted with 165 EV of 244 human lular space, hemoglobin complex, and blood microparticle proteins which were found in the HIV+ EVs (67.6%) (see (𝑃 < 0.001, Table 9), GO ontologies for molecular function Supplementary Material 2, including PMIDs for references). were heparinbinding,iongatedactivity, andoxygentrans- Functional analysis of the control EVs are listed in Table 8. porter activity, and the most significant biological processes The major sites of expression were cervicovaginal uid fl , found were response to yeast, defense response to fungus, neutrophils, and gastric juice (𝑃<0.0001). The most signif- macrophage chemotaxis, negative regulation of growth of icant ontologies were molecular function of the proteins and symbiont in host, oxygen transport, and hydrogen peroxide defense/immunity protein activity and principal biological catabolic process. Con/ml E6 Con/ml E6 Int Int 10 Advances in Virology Cytoplasm 32.1 ∗∗ 75.4 ∗∗ Endothelium Plasma 67.9 ∗∗ Nucleus 32.4 ∗∗ Liver 65.5 ∗∗ Plasma mem ∗∗ 19.3 Kidney ∗∗ 57.8 Exosome 11.2 ∗∗ Lung 56.3 ∗∗ Lysosome 8.9 ∗∗ ∗∗ Serum 44.1 0 5 10 15 20 25 30 35 0 20406080 Percentage of proteins Percentage of proteins 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. Table 8: Functional enrichment analysis of control EV proteins. Genes in the Genes in the Bkg. Percentage of Corrected𝑃 value Fold enrichment database database genes (BH FDR ) Site of expression Cervicovaginal uid fl 16 544 12.0 4.2 2.59𝐸−06 Neutrophils 13 392 9.7 4.8 6.68𝐸−06 Gastric juice 9 222 6.7 6.1 4𝐸−05 Molecular function Defense/immunity protein activity 5 52 3.7 15.7 3.96𝐸−05 Biological process Immune response 13 561 9.8 3.4 0.00026 Signal transduction 43 3907 32.5 1.5 0.0026 Cell communication 41 3687 31.1 1.5 0.0028 Antigen presentation 1 1 0.7 134.4 0.0073 4. Discussion HIV+,1,932,and HIV−,only37.TEManalysisofHIV+and HIV− urine showed pleiotropic membrane bound vesicles in This is the rfi st report of the detection of urinary EVs both groups’ urine samples and NTA analysis showed parti- containing HIV and human proteins from HIV+ patients by cles ranging in size from 50 nm to 300 nm in both groups, mass spectrometry and western blot. EVs provide intercel- although the HIV+ samples had signicfi antly more particles lular communication to cells through the delivery of their than uninfected samples. Other studies have found increased cargo, nucleic acids, miRNAs, and proteins, to recipient cells numbers of EVs in the plasma of HIV+ patients [43, 49]. reviewed in [3]. Previous studies have found EVs in plasma of Proteins from both the HIV+ and HIV− individuals were sig- HIV+ patients but did not describe HIV or human proteins nicfi antlyassociatedwithexosomalproteins,furthersubstan- within them. Others have described EVs containing HIV tiating our hypothesis that urine from HIV+ patients contains proteins but these results were from in vitro HIV infected cell EVs (Table 10). eTh FunRich analysis of the sites of expression cultures and not from HIV+ patients [18, 20, 22, 23, 36, 38– showed that asignicfi antnumberofproteinswereassociated 47]. This study details both the HIV and human proteins with the endothelium, plasma, serum, kidney, liver, and lung. found in urinary EVs from HIV+ patients. These findings suggest that EVs from HIV+ patients may be According to the International Society for Extracellular filtered from these sites and concentrated in urine. Vesicles (ISEV), the minimal requirements for EVs or their HIV has previously been detected in the urine of HIV+ presence in samples includes the simultaneous detection of patients; however, it was shown that HIV virions are associ- transmembrane proteins and cytosolic proteins with mem- ated with cell pellets and not in centrifuged urine [50, 51]. brane/receptor binding abilities, while major cell organelles p24 is found in replicative HIV infectious virions but was are absent [48]. LC/MS/MS analysis identified these proteins not found in twenty-six of our HIV+ samples by ELISA and functional enrichment analysis determined a significant and only five of thirty-five HIV+ EV urine samples had number which were of exosomal origin in both the EVs in detectable p24 by LC/MS/MS analysis. p24 in urine pellets Site of expression Cellular component Advances in Virology 11 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 12 Advances in Virology Table 9: Continued. Gene 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 Bkg. Percentage of Corrected𝑃 value Fold enrichment data set database genes (Bonferroni method) Site of expression Urine 31 3202 51.7 3.0 6.85𝐸−07 Cervicovaginal uid fl 12 544 20.0 7.2 5.33𝐸−05 Neutrophils 9 392 15.0 7.7 1.56𝐸−03 032403 BALF4 glypep 4 43 6.7 35.0 3.53𝐸−03 Neutrophil 19 1979 31.7 3.0 3.67𝐸−03 Monocyte 23 2786 38.3 2.6 3.90𝐸−03 Cellular component Extracellular 22 1808 37.9 3.1 4.61𝐸−05 Stored secretory granule 3 19 5.2 51.0 3.27𝐸−03 Lysosome 17 1609 29.3 2.8 7.73𝐸−03 Extracellular space 8 399 13.8 5.6 1.05𝐸−02 Exosomes 19 2001 32.8 2.5 1.14𝐸−02 Azurophil granule 2 6 3.4 108.9 1.35𝐸−02 is derived from mononuclear cells but was found in only 3 203 and 311 days, after the rst fi sample that had similar results. of 80 analyzed samples [51]. This represents a low sensitivity, The identification of HIV proteins in urinary EVs may be a primarily because the HIV-1 p24 protein is not always present potential noninvasive diagnostic tool to monitor HIV disease during advanced stages of HIV infection. To further confirm states as well as treatment efficacy. that these HIV proteins were from EVs, we tested the filtrate Different proteins and pathways were found in EVs from from ultracentrifugation (MW cutoff 100,000 kD) of HIV- (1)CD4+Tcell> 300 versus<300 and (2) VLs< 200 versus positive urine, and no HIV proteins were present. We did >200 copies. It is interesting that EVs from HIV+ patients not, however, perform an HIV infectivity assay, MAGI, on the withlowVLsandhighCD4+Tcells,usuallyindicativeof isolated urinary EVs, and thus cannot be totally cond fi ent that better health, had more proteins detected than EVs from HIV virions were not present in the EVs. HIV proteins in uri- highVLsandlowCD4+Tcells(highVLs=2486vrslow nary EVs may be the result of a nonproductive HIV infection = 15028; low CD4+ T cells = 2115 versus high CD4+ = in the kidney [52–56] and/or EVs filtered from blood [21, 49, 15761). es Th e groups also had overlapping pathway results; 57]. The type of HIV protein in the EVs remained relatively however, proteins from high VLs and low CD4+ T cells did constant as demonstrated by the resampling of two patients, not have similar pathway results. Further comparison and Advances in Virology 13 analysis of the EV protein profile between the low VL/high One of the limitations of this study was a small sample CD4+ T cells and high VL/low CD4+ T cells may reveal size of specific HIV syndromes such as comorbidities, AIDS, more mechanisms involved in the evolving pathology of HIV HIV-associated nephropathy, and HIV-associated dementia infection. as well as patients on or na¨ıve to antiretroviral therapy. Proteins contained in EVs can both enhance and inhibit Increasing the numbers of HIV+ patients in these categories host responses from innate, inflammatory, and adaptive may allow us to determine whether specific HIV proteins as reactions. Proteins from HIV+ patients showed a predom- well as human proteins in urinary EVs could be associated inantly immunosuppressive profile. IL10 is a Th2 cytokine with these conditions. Future studies will also quantify the that downregulates macrophage function and inhibits T cell amount of HIV proteins as well as human proteins to deter- proliferation while IL6 can stimulate IL10 production and mine if a correlation exists between different HIV conditions inhibit the effects of TNF- 𝛼 and IL1. Both these cytokines and the amount of proteins detected. were present in the EVs from HIV+ patients while TNF-𝛼 HIV infection is usually detected by antibodies to HIV and IL1 were not detected suggesting an immunomodulatory and can take up to three months to develop or by measuring eect ff may be elicited by the EVs. Other immune downregu- VLs in blood whereas we can detect HIV-1 proteins in lating factors, IDO, HOXB4, HGF, and TGF𝛽1, were found. urinary EVs. In summary, urinary proteins in EVs from IDO [58], HLA-G [59], and HGF [60] can inhibit natural HIV+ patients may allow a noninvasive method to (1) rapidly killer cell activation which was one of the top biological screen forinfection andidenticfi ationofpatientseligiblefor processes found in the pathway analysis of the EV proteins antiretroviral treatment (ART); (2) monitor ART treatment in patients with high CD4+ T cells and low VLs. TGF𝛽-1, an efficacy; and (3) diagnose HIV comorbidities. inhibitor of immune function, is induced by HIV Tat [61] and isamediatorofimmunesuppressionin HIVinfection [62– Conflicts of Interest 64]. These proteins were found in EVs from HIV+ patients while proinflammatory cytokines were not. New studies show eTh authors declare that they have no conflicts of interest. that HIV+ nonprogressors have lower plasma TGF𝛽-1 and IL10 than patients with progressive disease [65] and it is Acknowledgments possible that EVs may sequester TGF𝛽-1 and IL10 and remove them from circulation. The presence of over 16 different The authors acknowledge Jane Chu and Mahfuz Khan of MHC Class I and II antigens in the EVs from HIV+ patients Morehouse School of Medicine for technical assistance and may support the hypothesis that this mechanism is used Dr. Douglas Paulsen for his support and editorial sugges- by intracellular pathogens to evade the immune response tions. This study was supported by the National Center for by decreasing cytotoxic T cell activity [66]. Herpes Simplex Advancing Translational Sciences of the National Institutes Virus-1 binds to HLA-DR inhibiting antigen presentation that of Health under Award no. UL1TR000454. Other funds were leads to immune evasion [67]. Future studies should focus on received from the Minority Biomedical Research Support the correlation of the concentration of these factors to HIV+ (MBRS) of the Research Initiative for Scientific Advancement patients’ clinical status. (RISE) Program 5R25GM058268 funded by NIGMS and In this study, we showed that structural, regulatory, and NIH Research Endowment S21MD000101 funded by the accessory HIV proteins could be detected in urinary EVs of National Institute on Minority Health and Health Disparities HIV+ patients. Our WB analysis using polyclonal and mon- (NIH/NIMHD). The authors also acknowledge the Research oclonal antibodies conrfi med the presence of HIV proteins Centers in Minority Institutions (RCMI) G12 funded by in the EVs from HIV+ patients. eTh most prevalent protein the NIH/NIMHD, #8G12MD0076202. MEB Core facility wasHIV Nef. EVsfromboth in vitro and patient samples was constructed with support from the Research Facili- have been previously reviewed in [6]. HIV Nef induces an ties Improvement Grant C06 RR18386 from NIH/NCRR. alternative pathway for TNF induction utilizing Notch-1, The newly renovated Core Resources space was funded ADAM17, and Rab4+, all found in EVs from HIV+ patients, by G20 RR031196 from NIH/NCRR. The R-CENTER was which leads to high plasma TNF levels [68]. Whether the funded by Grant no. U54MD007588 from NIH/NIMHD and isolation of these factors in EVs represents a diminishing or NIH/NCRR 5P20R R0111044 pilot for study support. enhancement of TNF production remains to be examined. The HIV Human Interaction database found significant Supplementary Materials interactions between HIV Nef, Vpr, Vif, and Vpu and human proteins. Serine/threonine protein kinases are important in Supplementary . Top 10 biological function pathways using T cell receptor signaling [69]. es Th e kinases as well as CD4 Pathway Studio 11 Mammal Plus, Elsevier, Inc., for HIV+ EV and MHC antigens were found in EVs from the HIV+ proteins from HIV+ patients with (1) CD4+ T cells greater samples; however, further studies are needed to determine the than 300, (2) CD4+ T cells less than 300, (3) viral loads less mechanisms involved with EV function in HIV infections. than 200 copies, and (4) viral loads greater than 200 copies. Cell adhesion molecules, ICAM, VCAM, and PECAM, were also found in the EVs from patients. Others have reported Supplementary . 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Detection of HIV-1 and Human Proteins in Urinary Extracellular Vesicles from HIV

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Copyright © 2018 Samuel I. Anyanwu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Advances in Virology Volume 2018, Article ID 7863412, 16 pages https://doi.org/10.1155/2018/7863412 Research Article Detection of HIV-1 and Human Proteins in Urinary Extracellular Vesicles from HIV+ Patients 1 1 1 Samuel I. Anyanwu, Akins Doherty, Michael D. Powell, 2 1 3 Chamberlain Obialo, Ming B. Huang, Alexander Quarshie , 1 2 1 Claudette Mitchell, Khalid Bashir, and Gale W. Newman Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, GA, USA Department of Medicine, Morehouse School of Medicine, Atlanta, GA, USA 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 Copyright © 2018 Samuel I. Anyanwu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Extracellular vesicles (EVs) are membrane bound, secreted by cells, and detected in bodily uids, fl 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 )andHIV− (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. 1. Introduction only accounts for 0.01% of urine components; however, in certain disease states, the protein content and EV numbers Extracellular vesicles (EVs) are membrane bound vesicles, can increase in urine [12–16]. The glomerular capsule filters between 30 nm and 1𝜇minsize, aresecretedintoblood, bloodthatispassedintotherenaltubuleandaccounts for urine, saliva, semen, and other bodily uids, fl and have been thirty percent of the urinary protein content [14–16]. eTh suggested as a potential source of biomarkers for disease pro- remaining seventy percent of proteins in urine is derived from gression [1, 2]. These EVs, microparticles and/or exosomes, the kidney [17], and thus, urinary EVs are comprised of both aresecretedbycellsnormally orwhiletheyareundergoing renal and efferent components. stress or apoptosis [3] and contain proteins, mRNA, and HIV proteins are detected in EVs of HIV+ patients and miRNA [4] that are involved in cell to cell communication, HIV Nef is the most prevalent protein found [18–21]. Other transfer of antigens to cells, and intracellular communication. reports of HIV proteins in EVs are from in vitro transfected EVs are described in cancer disease pathogenesis [5] in HIV or HIV infected cultured cells and are not from HIV+ patient infection [6], other viral infections [7], and other disease samples [6, 18, 19, 22, 23]. states such as cardiovascular, renal, liver, and metabolic Biomarkers in urinary EVs are suggested for use in the disease [8–11]. diagnosis of many disease states [12, 13, 24–30]. The objectives EVsfromurine areanattractivenoninvasive sourcefor of this study were to determine the differences in proteins biomarkers of diseases [12, 13]. In healthy individuals, protein from urinary EVs from HIV+ patients and HIV− individuals 2 Advances in Virology Table 1: Patient demographics. a JEOL 1200EX transmission electron microscope (JEOL, Peabody, MA). HIV-positive HIV-negative Characteristics (𝑁 =35) (𝑁 =12) .. Nanoparticle Tracking Analysis (NTA). Urine samples Age (median± IQR) 41.5± 14.25 59± 18 from HIV-negative (𝑛=8 )andpositive individuals(𝑛= Sex (𝑛,%) 11), 15 ml, were centrifuged at 300×gfor 10minat4 C Male 25 (71.4%) 7 (58.3%) to remove cell debris. eTh supernatant was collected and Female 10 (28.6%) 5 (41.7%) centrifuged at 16,500×gfor20minat4 Candthesuper- Race (𝑛,%) natant collected and ultracentrifuged at 120,000×gat4 C for 1.5 hr. eTh pellet was resuspended in 500 𝜇lofPBS. African American/Black 28 (80%) 12 (100%) eTh size and quanticfi ation of the EVs were analyzed using White 7 (20%) - the NanoSight NS500 (NanoSight NTA 2.3 Nanoparticle Hispanic - - Tracking and Analysis Release version build 0025). Particles Asian - - were automaticallytracked andsized basedonBrownian Viral loads (copies/ml) (median motion and the diffusion coefficient. The NTA measurement 50±0- ± IQR) ∘ conditions were temperature 21.0 +/− 0.5 C, viscosity 0.99 CD4+ T cell (cells/𝜇l) (median ± +/− 0.01 cP, frames per second 24.99–25, and measurement 66.5± 46.5 - IQR) time 30 s. The detection threshold was similar in all samples. Antiretroviral therapy (𝑛, %) 34 (97.1%) - Two recordings were performed for each sample. .. Mass Spectrometry Analysis. Thirty-five (35) HIV+ and twelve (12) HIV− EV samples were lysed and trypsinized using proteomics and mass spectrometry. eTh analysis of and the sequence of peptides was determined by tandem more patient samples could identify specific EV urinary proteins as biomarkers of HIV infection, treatment ecffi acy, mass spectrometry (LC/MS/MS), using an LTQ Ion Trap Mass Spectrometer (Thermo Fischer Scientific, Waltham, and/or disease progression. MA). Peptides were rfi st reduced in DTT 10 mM at 56 C foratleast 30minand alkylatedwith15mMiodoaceticacid 2. Methods for 30 min at room temperature in the dark. Samples were then digested with mass spectrometry grade trypsin 20 ng/𝜇l .. Sample Collection. Urine was collected from thirty-five for 4 hours at 37 C. Just before analysis, the sample was (35) HIV+ patients and twelve (12) HIV− individuals in sterile acidified by the addition of formic acid to 0.1%. Peptides collection cups. eTh subjects were recruited from clinics in were separated by reverse phase HPLC (Agilent) on a 0.5× the metropolitan Atlanta area, GA. Patient demographics are 75 mm C-18 column (Michrom) at a flow rate of 500 nl/min described in Table 1. eTh study was approved by the Institu- using a linear gradient of acetonitrile (5–35%) over 100 min. tional Review Board of Morehouse School of Medicine and Ions were directly introduced by nanospray and spectra written informed consent was obtained from all participants. were collected using Xcalibur 2.0 software using an intensity thresholdof200counts.Theresulting spectrawereanalyzed .. EV Isolation. Urine samples were centrifuged at 1000×g using Bioworks 1.1 software to search a hybrid Human-HIV to remove cells and sediment then frozen at−80 C. Samples, database created from the complete nonredundant peptide 4 ml, were thawed and the EVs isolated followed by centrifu- database from NCBI. eTh threshold for inclusion in the gal filtration using Amicon Ultra-4 100 kDa centrifugal filter search is a minimal S/N ratio of 3. False discovery rates were unit (Millipore, Billerica, MA), at 3000×gfor15minutesat determined and set based on the control HIV− samples. An 4 C. The retentate, containing EVs, was collected from the initial protein identification list was generated from matches top of the filter and resuspended in 200 𝜇lphosphate bueff red with an Xcorr score versus charge state of 1.0 (+1) 1.5 (+2) and saline (PBS) for use in the transmission electron microscopy 1.7 (+3) and consensus scores greater than 10.0. and tandem mass spectrometry (LC/MS/MS) analysis. Bioinformatics techniques for analysis of HIV EV pro- teins were used on the LC/MS/MS detected proteins [31]. .. Transmission Electron Microscopy Analysis. Transmis- Functional enrichment analysis was performed using Fun- sion electron microscopy (TEM) was used to identify EVs in Rich (Functional Enrichment analysis tool, http://funrich two HIV-1 positive and two HIV-1 negative samples. Urinary .org/index.html) [32] against a human database to detect pro- EVswerefixedin2.5%glutaraldehydein0.1Mcacodylate teins involved in biological processes, cellular components, buffer for 2 hours at 4 Cfollowedby2washeswith 0.1M sites of expression, and biological pathways. Only processes cacodylate buffer, 5 minutes each. Samples were stained with with a𝑃 value< 0.05, using the Benjamini-Hochberg False 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 hour at Discovery rate, were reported. eTh human proteins detected 4 C followed by 2 washes with the cacodylate bueff r and 3 were compared to the top 100 EV proteins in ExoCarta washes with deionized water, 5 minutes each. Samples were (http://exocarta.org/exosome markers new) [33, 34], sixty subsequently stained with 0.5% aqueous uranyl acetate for EV proteins in the EV array [35], and proteins identified in 2 hours at room temperature and subsequently viewed with EVs from HIV infected lymphocytic cells [36]. Advances in Virology 3 Pathway analysis comparing HIV+ samples with CD4+ Rev, reverse transcriptase, Tat, Vif, Vpr, and Vpu. All HIV+ T cells greater than 300 (𝑛=15 ) to those with less than urine samples (𝑛=35 ) contained at least one HIV-1 protein in 500 (𝑛=15 )andHIVhighVL,greaterthan200 copies EVs, while no HIV proteins were found in the HIV− samples (𝑛=10 ), compared to HIV low viral loads, less than 200 (𝑛=12 ) (Table 3). HIV-1 Nef was detected in twenty-six copies (𝑛=10 ), was done using Pathway Studio version of thirty-five (26 of 35) (74.3%) HIV+ urine samples. Three 11.4 Mammal Plus (Elsevier, Inc., Atlanta, GA). Gene Set (3) patients’ urine samples, #173, #174, and #196, were tested Enrichment Analysis (GSEA) was used to identify the top 10 203, 311, and 35 days, respectively, aer ft their first EV sample curated pathways for the proteins in the each of the patient was analyzed. No difference in the HIV proteins detected in groups. No comparisons were done between patients not on sample #196, 35 days aeft r his previous sample, was found. ART or undergoing ART because there was only one patient #173’s sample,tested203 days aeft r thefirstanalysis, hada not on ART. similar profile, except that Rev and Tat were not detected. In e Th HIV proteins, Nef, Vpr, Vpu, and Vif, were searched addition, #174’s EVs examined 311 days aer ft the first sampling using the HIV-1 Human Interaction database (https://www foundRev andRTmissingfromtheprofile. .ncbi.nlm.nih.gov/genome/viruses/retroviruses/hiv-1/inter- HIV p24 antigen was only detected in ve fi of thirty-ve fi actions/). This database contains all the known, published (5 of the 35 patient) (14%) samples by LC/MS/MS, but of the interactions of HIV-1 gene products with human proteins twenty-six (26) HIV+ and eleven (11) HIV-negative samples [37]. Proteins from the search were compared to the human tested by ELISA, no p24 was detected. There was no statistical proteins detected in the HIV EVs. correlation of the number of HIV proteins detected with CD4+ T cell counts, viral loads, or ART therapy. .. Western Blot Analysis. To validate the presence of HIV Validation by WB analysis using polyclonal pooled proteins in urinary EVs, western blot analysis (WB) was patient serum and monoclonal antibodies against HIV Nef performedontwenty(20)randomlyselectedHIV+and andHIV p24indicatedthepresenceofHIV proteins.Figure1 three (3) HIV− control urine samples. Recombinant HIV-1 is a WB using polyclonal pooled HIV+ serum used as the Nef and HIV-1 p24 were used as positive controls, while detection antibody. All the HIV+ patient samples contained HIV-negative urine and HIV-positive filtrate were used HIV-1 proteins and the top panel shows patient samples as negative controls. Samples were heated at 85 Cfor two reacting to anti-HIV Nef. HIV+ urine samples, 7 of 9 (77.7%), minutes in a tris-glycine SDS sample buffer, were loaded into showed HIV-1 Nef bands at 27 kD. a 4–20% TGX gradient gel (Bio-Rad, Hercules, CA), and run for 50 mins at 200 V. A semidry transfer unit (Hoefer .. TEM and NTA Analysis of EVs. TEManalysisofurine Scientific, Holliston, MA) was used to transfer the separated from HIV+ patients showed multiple EVs, ranging in size proteins onto a PVDF membrane (Bio-Rad) at 15 V for from 50 nm to 300 nm (Figure 2(a)), while two HIV-negative 50 mins. eTh filter was blocked for nonspecific binding controls had fewer EVs present (Figure 2(b)). NTA analysis using 5% nonfat dry milk in 1x tris buffered saline (TBS) showed that there were signicfi antly more EVs from HIV+ with Tween 20. The membrane was incubated overnight in patients than healthy controls,4.96±0.0733 and3.69±0.075, pooled plasma from twenty HIV+ patients as the primary respectively (𝑃<0.05). No significant differences were found antibody at a 1 : 500 dilution and rabbit anti-human IgG in the size of the EVs, 110–227 nm for HIV-negative donors conjugated HRP antibody (1 : 1000, Bio-Rad, Hercules, CA) and 54–448 nm HIV+ samples. Representative Nanosight wasusedassecondaryantibody.Super Signal West Femto analyses for HIV-negative and HIV+ urine samples are (Thermo Fischer Scientific, Waltham, MA) was used as a showninFigure3. chemiluminescent substrate for detection. eTh membrane wasdeveloped andimagedusingtheLAS 4000biomolecular .. Human Proteins in HIV+ and Negative EV Urine Sam- imager (GE Healthcare Life Sciences, Pittsburgh, PA). ples. EV proteins from the HIV+ patients, 14,475, which Recombinant HIV-1 Nef and p24 WB analyses were detected enteredintoFunRich,functionalenrichmentanalysisso-ft using anti-Nef and p24 monoclonal antibodies (1 : 500, EMD ware, showed 29.44% or 1,932 proteins were associated with Millipore, Billerica, MA) and anti-mouse IgG conjugated exosomes (Table 4). es Th e EV identified proteins were com- HRP antibodies (1 : 1000, Bio-Rad, Hercules, CA) were used. pared to top 100 EV proteins in the ExoCarta database with 83% matching (http://exocarta.org/exosome markers new) .. HIV p ELISA. Twenty-six (26) HIV+ and eleven (11) [33], 22 EV proteins in the EV array [35] were similar, and HIV− urine samples were tested for the presence of HIV p24 7 of 14 EV proteins identified in exosomes from HIV infected by ELISA (ImmunoDX, Woburn, MA). lymphocytes [36] were found and are highlighted in Table 4. Exosomal proteins found in the control samples are listed in Table 5. 3. Results eG Th O resultsoftheFunRichanalysisoftheEVs .. HIV Proteins Are Present in Urinary EVs of HIV-Positive from the HIV+ samples are summarized in Table 6 and Patients. LC/MS/MS mass spectrometry HIV EV protein Figure 4. The top vfi e ( 𝑃 < 0.01) EV sites of expression results are presented in Table 2. Urinary EV proteins meeting were endothelial cells, plasma, liver, serum, and kidney and the false discovery rate and Xcorr score criteria as HIV-1 themostsignicfi antcellularcomponentswerelysosomes, proteins included Nef, Gag, Pol, Protease, gp120, gp160, gp41, exosomes, membranes, plasma membranes, the nucleus, and 4 Advances in Virology Table 2: LC/MS/MS analysis of EV HIV proteins. Accession # AAs MW [kDa] Calc. pI Description Σ Coverage Σ# peptides Score A0 Coverage A0 # peptides A0 gi38491705 192 22.7 10.1 Vif protein [human immunodeficiency virus 1] 13.54 12 9.22 13.54 4 gi73913089 104 11.7 10.1 Gag protein [human immunodeficiency virus 1] 14.42 15 6.27 14.42 4 gi58374258 869 98.1 8.8 Envelope glycoprotein [human immunodeficiency virus 1] 1.5 5 5.63 1.5 3 gi183197180 404 45.8 8.4 Pol protein [human immunodeficiency virus 1] 3.47 3 5.27 3.47 3 gi255984636 160 18.1 5.3 Reverse transcriptase [human immunodeficiency virus 1] 7.5 4 4.80 7.5 2 gi256012108 114 13.5 5.7 Nef protein [human immunodeficiency virus 1] 14.04 3 4.53 14.04 2 gi9756252 524 60.4 8.7 Pol precursor [human immunodeficiency virus 1] 4.01 2 4.46 4.01 2 gi67082579 191 22.3 9.4 Reverse transcriptase [human immunodeficiency virus 1] 10.47 2 4.43 10.47 2 gi2290009 852 96.7 8.5 Envelope glycoprotein [human immunodeficiency virus 1] 7.16 11 4.33 5.87 3 gi167886806 25 2.7 8.7 Rev protein [human immunodeficiency virus 1] 56 4 4.29 56 2 gi23344577 99 10.6 9.4 Protease [human immunodeficiency virus 1] 12.12 5 4.36 12.12 2 gi4324808 1437 161.9 8.3 Gag-pol polyprotein [human immunodeficiency virus 1] 2.51 7 4.05 1.6 2 gi222533599 73 8.0 9.1 Env C2V3 protein [human immunodeficiency virus 1] 23.29 4 3.85 23.29 2 gi71060450 206 23.7 6.3 Negative factor [human immunodeficiency virus 1] 4.85 4 3.84 4.85 2 gi37935985 85 10.3 4.8 Vpu protein [human immunodeficiency virus 1] 11.76 4 3.84 11.76 2 gi108860432 870 98.8 8.5 gp160 [human immunodeficiency virus 1] 3.33 2 3.74 3.33 2 gi114801226 209 24.4 10.1 Tat protein [human immunodeficiency virus 1] 5.26 2 3.70 5.26 2 gi22596451 341 38.6 8.0 Truncated envelope glycoprotein [human immunodeficiency virus 1] 3.81 4 3.68 3.81 2 gi183200570 342 38.7 9.2 Truncated pol protein [human immunodeficiency virus 1] 3.51 2 3.68 3.51 2 gi34786230 176 19.8 9.6 gp120 protein [human immunodeficiency virus 1] 10.8 3 3.60 10.8 3 gi1002239 104 11.5 8.9 Envelope glycoprotein, v3 region [human immunodeficiency virus 1] 18.27 2 3.54 18.27 2 gi77168129 95 11.3 7.6 Vpr protein [human immunodeficiency virus 1] 13.68 3 3.40 13.68 2 gi222532593 129 14.7 8.4 Gag p17 protein [human immunodeficiency virus 1] 8.53 3 2.61 8.53 1 gi219688191 132 14.9 10.2 Matrix protein [human immunodeficiency virus 1] 9.09 6 2.56 9.09 1 gi255687141 288 32.1 7.7 Integrase [human immunodeficiency virus 1] 9.03 1 2.52 9.03 1 gi222532161 132 15.0 9.5 Gag p17 protein [human immunodeficiency virus 1] 9.09 6 2.45 9.09 1 gi37934078 573 65.0 9.0 Gag-pol fusion polyprotein [human immunodeficiency virus 1] 2.79 2 2.42 2.79 2 gi405003 207 23.1 7.7 gp120 [human immunodeficiency virus 1] 12.08 1 2.41 12.08 1 gi54792352 213 23.7 5.6 Gag polyprotein [human immunodeficiency virus 1] 9.39 2 2.34 9.39 2 gi3885826 132 14.9 9.6 p17 matrix [human immunodeficiency virus 1] 11.36 2 2.31 11.36 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. Advances in Virology 5 Table 3: Presence of HIV-1 proteins in HIV+ patient urinary EVs. Viral load CD4 ID ART AIDS Nef Gag Pol Protease Rev RT Tat Vif p1 p24 p17 Poly Vpu Env Vpr Vif cells/ul copies/ml 22 No XX X X X X X X X X 27 Yes AIDS <50 X 28 Yes AIDS 280100 XX X X X 30 Yes AIDS >10000 XX X X X X X X XX <20 41 Yes 29187 XX X 46 Yes <50 XX X X 45 Yes 400 XX X X X 48 Yes 4974 XX XX 51 NA XX X X X NA 52 Yes 51 XX X X X X X X 61 <50 XX 62 Yes AIDS <50 X 63 No 2023 X 65 No NA X NA 66 No NA X NA 67 Yes 75 X 68 No NA X NA 69 Yes <50 X 70 Yes <50 X 71 Yes <50 456 X 74 NA X NA 86 Yes <75 XX X X 103 Yes AIDS 150 XX X 104 Yes AIDS 77 313 XX 108 Yes AIDS <50 XX X 110 Yes <50 XX 111 Yes AIDS <50 XX 112 Yes AIDS >200 581 XX 142 Yes <50 XX X X X X X X 173-1 Yes <50 XX X X X X X X X X 173-2 Yes <50 XX X X X X 174-1 Yes 48 315 XX X X X X 174-2 Yes 48 XX X X 196-1 Yes AIDS <50 XX X X X X X X X X X X X 196-2 Yes AIDS <50 XX X X X X X X X X X 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 consens us scores greater than 10.0; NA = not available. the cytoplasm(𝑃<0.01) (Figure 4). The top vfi e ontologies T cells less than 300, 15,028 proteins from patients with low (Table 6) were protein serine/threonine kinase activity, cat- VL, and 2486 from patients with high VLs. Pathway analysis alytic activity, GTPase activator activity, guanyl-nucleoside was similar between EV proteins from patients with greater exchange factor activity, and cell adhesion molecule activity than 300 CD4+ T cells and low VLs and different between (𝑃 < 0.0001), the top biological process was regulation of the low CD4+ T cells and high VLs (summarized in Table 7). nucleobase, nucleoside, and nucleic acid (𝑃< 0,0001), and the The pathways found are detailed in Supplementary Material most prominent biological pathway was integrin cell surface 1. Interleukin proteins detected were IL10, IL10RA, IL16, interactions (𝑃<0.03). IL17RC, IL18, IL18BP, IL1RAP, IL1RL2, IL1RN, IL33, IL4I1, LC/MS/MS identified 15,571 proteins in EVs from HIV+ IL6, and IL6ST. Immunomodulatory molecules, HOXB4, patients with CD4+ T cells greater than 300, 2,115 from CD4+ CD81,CD9,TGF-𝛽1, IDO, Notch1, ADAM17, Rab4, and HGF, 6 Advances in Virology Table 4: Exosomal proteins found in urinary EVs from HIV+ patients. Genes in our analysis Genes in the FunRich database Percentage of genes Fold enrichment Exosomal proteins 1932 2001 29.44 2.11 A1BG, AM, AARS, ABCA7, ABCB1, ABCB11, ABCB6, ABCC1, ABCC11, ABCC9, ABCG2, ABHD8, ACAA2, ACAT1, ACAT2, ACE, ACE2, ACLY, ACO1, ACOT11, ACP2, ACSL3, ACSL4, ACSM1, ACTA1, ACTA2, ACTB, ACTBL2, ACTC1, ACTG1, ACTL6A, ACTN1, ACTN2, ACTN4, ACTR1A, ACTR1B, ACTR2, ACTR3, ACY1, ACY3, ADAM10, ADAMTS3, ADCY1, ADH5, ADH6, ADK, ADSL, AEBP1, AGAP2, AGR2, AGR3, AGRN, AGT, AHCTF1, AHCYL1, AHNAK, AHSA1, AHSG, AK1, AK2, AKAP9, AKR1A1, AKR1B10, ALAD, ALB, ALCAM, ALDH16A1, ALDH1A1, ALDH1L1, ALDH2, ALDH3B1, ALDH8A1, ALDOA, ALDOB, ALDOC, ALK, ALOX12, ALPL, ALPP, ALYREF, AMBP, AMN, ANGPT1, ANGPTL1, ANGPTL4, ANKFY1, ANKRD11, ANO1, ANO6, ANPEP, ANXA, ANXA, ANXA13, ANXA3, ANXA, ANXA, ANXA7, AOX1, AP1M1, AP2A1, AP2A2, AP2M1, AP4M1, APAF1, APLP2, APOA1, APOA2, APOB, APOD, APOE, APOL1, APP, APPL1, APPL2, APRT, AQP2, ARF5, ARFIP1, ARHGAP1, ARHGAP23, ARHGDIA, ARHGDIB, ARHGEF12, ARHGEF18, ARL15, ARL3, ARL8B, ARMC3, ARMC9, ARPC1A, ARPC1B, ARPC2, ARPC3, ARPC5, ARRDC1,ARSE,ARSF,ARVCF,ASAH1,ASB6, ASL,ASNA1, ASNS,ATAD2,ATIC, ATPA, ATP1A2, ATP1A3, ATP2B1, ATP2B2, ATP2B4, ATP4A, ATP5A1, ATP5B, ATP5L, ATP6AP1, ATP6AP2, ATP6V0A1, ATP6V0A4, ATP6V0C, ATP6V0D1, ATP6V0D2, ATP6V1A, ATP6V1B1, ATP6V1C1, ATP6V1C2, ATP6V1D, ATP6V1E1, ATP6V1H, ATRN, AUP1, AZGP1, AZU1, B2M, B3GAT3, B4GALT1, B4GALT3, BAIAP2, BAIAP2L1, BASP1, BAZ1B, BCAM, BCR, BDH2, BGN, BHLHB9, BHMT, BHMT2, BLMH, BLOC1S5, BLVRA, BLVRB, BMP3, BPI, BPIFB1, BPTF, BRI3BP, BROX, BSG,BTG2, BTN1A1,C11orf52,C11orf54,C16orf80,C16orf89,C17orf80, C19orf18,C1GALT1C1, C1orf116, C1QC, C1QTNF1, C1QTNF3, C1R, C2orf16, C3, C4BPA, C5, C9, CAB39L, CACNA2D1, CACYBP, CAD, CALM1, CALML3, CALR, CAMK4, CAMP, CAND1, CANX, CAP1, CAPN1, CAPN2, CAPN5, CAPN7, CAPNS1, CAPS, CAPZA2, CAPZB, CARD11, CASP9, CAV1, CBR3, CC2D1A, CCDC105, CCDC132, CCL28, CCPG1, CCT, CCT, CCT4, CCT, CCT6A, CCT7, CCT8, CD101, CD14, CD163L1, CD19, CD2, CD22, CD274, CD2AP, CD300A, CD36, CD37, CD40, CD44, CD53, CD55, CD58, CD59, CD63, CD70, CD74, CD79B, CD80, CD81, CD9,CD97, CDC, CDC42BPA, CDC42BPB, CDH1, CDH17, CDHR2, CDHR5, CDK1, CDK5RAP2, CDKL1, CEACAM5, CELSR2, CEMIP, CEP250, CES2, CETP, CFD, CFH, CFI, CFL, CHGB, CHID1, CHMP1A, CHMP2B, CHMP4B, CHRDL2, CHST1, CHST14, CIB1, CKAP4, CKB, CLASP1, CLCA4, CLDN3, CLDN4, CLDN7, CLIC1, CLIC4, CLIC5, CLIC6, CLIP2, CLSTN1, CLTC, CLTCL1, CLU, CMPK1, CNDP2, CNKSR2, CNTLN, COASY, COBLL1, COL12A1, COL15A1, COL18A1, COL6A1, COL6A2, COL6A3, COLEC10, COLGALT1, COMT, COPA, COPB1, COPB2, COPS8, CORO1A, CORO1B, COX4I1, COX5B, CP, CPD, CPN2, CPNE1, CPNE3, CPNE5, CPNE8, CPVL, CR1, CR2, CRB2, CREB5, CRISPLD1, CRNN, CRTC2, CRYAB, CRYZ, CS, CSE1L, CSK, CSPG4, CSRP1, CST4, CSTB, CTDSPL, CTNNA1, CTNNB1, CTNND1, CTSB, 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, DDX11, DDX19A, 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, DNAJC7, DNHD1, DNM2, DNPH1, DOCK10, DOCK2, DOPEY2, DPEP1, DPP3, DPP4, DPYS, DPYSL2, DSC2, DSG2, DSG3, DSP, DSTN, DUOX2, DUSP26, DUT, DYNC1H1, DYNC2H1, DYSF, ECE1, ECH1, ECM1, EDIL3, EEA1, EEFA, EEF1A2, EEF1D, EEF1G, EEF, EFEMP1, EFEMP2, EGF, EGFR, EHD1, EHD2, EHD3, EHD, EIF2S1, EIF2S3, EIF3A, EIF3B, EIF3E, EIF3L, EIF4A1, EIF4A2, EIF4A3, EIF4E, EIF4G1, EIF4H, ELANE, EML5, ENO, ENO2, ENO3, ENPP3, ENPP4, ENPP6, ENTPD1, EPB41L2, EPCAM, EPHA2, EPHA5, EPHB1, EPHB2, EPHB3, EPHB4, EPHX2, EPN3, EPPK1, EPRS, EPS8, EPS8L1, EPS8L2, EPS8L3, ERAP1, ERBB2, ERMN, ERO1L, ERP44, ESD, ETFA, EVPL, EXOC4, EXOSC10, EXT2, EYS, EZR, F11, F11R, F5, F7, FABP1, FABP3, FAH, FAM129A, FAM129B, FAM151A, FAM208B, FAM209A, FAM20A, FAM20C, FAM49B, FAM65A, FAS, FASLG, FASN, FAT1, FAT2, FBL, FBP1, FBP2, FCGBP, FCN1, FCN2, FERMT3, 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, G6PD, GAA, GABRB2, GAL3ST4, GALK1, GALM, GALNT3, GANAB, GARS, GART, GATSL3, GBE1, GBP6, GCN1L1, GCNT2, GCNT3, GDF2, GDI, GDPD3, GEMIN4, GFPT1, GGCT, GGH, GGT1, GHITM, GIPC1, GK, GK2, GLB1, GLDC, GLG1, GLIPR2, GLO1, GLUD1, GLUL, GNA13, GNAI1, GNAI,GNAQ, GNAS, GNB, GNB, GNB2L1, GNB3, GNB4, GNB5, GNG12, GNPDA1, GNPTG, GOLGA4, GOLGA7, GOT1, GOT2, GPC1, GPC4, GPD1, GPI, GPM6A, GPR155, GPR64, GPR98, GPRASP1, GPRC5A, GPRC5B, GPT, GREB1, GRHPR, GRID1, GRIN1, GRK4, GSN, GSR, GSS, GSTA3, GSTCD, GSTK1, GSTO1, GSTP1, GUSB, H1FOO, H2AFY, H2AFY2, HADHA, HAPLN3,HAUS5,HBB,HBD,HBS1L,HDHD2, 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, HSP90AB1, HSP90B1, HSPA12A, HSPA13, HSPA1L, HSPA2, HSPA4, HSPA,HSPA6, HSPA , HSPA9, HSPB1, HSPB8, HSPD1, 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, IST1, ITFG3, ITGA1, ITGA2, ITGA2B, ITGA3, ITGA4, ITGA,ITGAL,ITGAV, ITGB,ITGB2,ITGB3,ITGB4,ITGB7,ITGB8,ITIH2,ITIH4,ITM2C, ITSN1,ITSN2,IVL, JADE2,JUP, KALRN, KCNG2,KHK, KIAA1324, KIF12, KIF15,KIF18B,KIF3A, KIF3B, KIF9,KIFC3,KL, KNG1, KPNB, KPRP, KRT1, KRT10, KRT12, KRT14, KRT15, KRT16, KRT17, KRT18, KRT19, KRT2, KRT20, KRT24, KRT25, KRT27, KRT28, KRT3, KRT5, KRT6C, KRT7, KRT73, KRT75, KRT76, KRT77, KRT78, KRT79, KRT8, KRT9, L1CAM, LAD1, LAMA3, LAMA4, LAMA5, LAMB2, LAMB3, LAMC1, LAMC2, LAMP1, LAMP2, LAMTOR3, LBP, LCK, LCP1, LDHA,LDHB,LEPRE1, LFNG,LGALS3, LGALSBP, LGALS4, LIMA1, LIN7A, LIN7C, LMAN1, LMAN2, LOXL4, LPO, LRP1, LRP1B, LRP2, LRP4, LRPPRC, LRRC15, LRRC16A, LRRC57, LRRK2, LRSAM1, LSP1, LSR, LTA4H, LTBP3, LTF, LUZP1, LYPLA2, MAGI3, MAL2, MAN1A1, MAN1A2, MAN2A1, MAP4K4, MAP7, MARCKSL1, MARK3, MARS, MARVELD2, MASP1, MASP2, MBD5, MBLAC2, MCAM, MCPH1, MDH1, MDH2, MEGF8, MEP1A, MEST, METRNL, MFGE , MFI2, MGAM, MGAT1, MGAT4A, MID2, MIF, MINK1, MLLT3, MLLT4, MME, MMP24, MMP25, MMRN1, MMRN2, MNDA, MOB1A, MOB1B, MOGS, MPO, MPP5, MPP6, MS4A1, MSH6, MSN, MSRA, MTA1, MTAP, MTCH2, MTHFD1, MTMR11, MTMR2, MUC13, MUC16, MUC4, MUM1L1, MVB12A, MVB12B, MVP, MX1, MXRA5, MXRA8, MYADM, MYH10, MYH11, MYH13, MYH14, MYH3, MYH8, MYH , MYL6B, MYO15A, MYO1B, MYO1C, MYO1D, MYO1E, MYO1G, MYO5B, MYO6, MYOF, N4BP2L2, NAA16, NAA50, NACA, NAGLU, NAMPT, NAP1L4, NAPA, NAPG, NAPRT, NAPSA, NARS, NBR1, NCALD, NCCRP1, NCKAP1, NCKAP1L, NCL, NCOA3, NCSTN, NDRG1, NDRG2, NEB, NEBL, NEDD4, NEDD4L, NEDD8, NEU1, NID1, NIN, NIPBL, NIT2, NKX61, NONO, NOTCH1, NOX3, NPC1, NPEPPS, NPHS1, NPHS2, NPM1, NPNT, NQO2, NT5C, NT5E, NUCB1, NUCB2, NUDT5, NUMA1, NXPE4, OLA1, OPTN, OR2A4, OS9, OSBPL1A, OXSR1, P2RX4, P4HB, PA2G4, PACSIN2, PACSIN3, PADI2, PAFAH1B1, PAFAH1B2, PAGE2, PAICS, PAM, PARD6B, PARP4, PBLD, PCBP1, PCDHGB5, PCK1, PCLO, PCNA, PCSK9, PCYOX1, PDCD2, PDCD5, PDCD6, PDCD6IP, PDDC1, PDE8A, PDIA2, PDIA3, PDIA4, PDIA6, PDLIM7, PDZK1, PEBP1, PECAM1, PEF1, PEPD, PEX1, PFAS, PFKL, PFKP, PGAM1, PGD, PGK, PGLYRP1, PGM1, PHB2, PHGDH, PI4KA, PIGR, PIK3C2A, PIK3C2B, PILRA, PIP, PIP4K2C, PKD1, PKD1L3, PKD2, PKHD1, 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, PMEL, PNP, PODXL, POFUT2, PON1, PON3, POTEE, POTEF, POTEI, POTEM, PPA1, PPARG, PPFIA2, PPIA, PPIB, PPL, PPM1L, PPP1CB, PPP1R7, PPP2CA, PPP2R1A, PPP2R1B, PRCP, PRDX, PRDX3, PRDX4, PRDX5, PRG4, PRKAR2A, PRKCA, PRKCD, PRKCH, PRKCI, PRKCZ, PRKDC, PRKRIP1, PRNP, PROM1, PROM2, PROS1, PROZ, PRRC2A, PRSS23, PRTN3, PSAP, PSAT1, PSMA2, PSMA3, PSMA5, PSMA7, PSMB1, PSMB3, PSMB4, PSMB5, PSMB6, PSMB8, PSMB9, PSMC2, 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,QDPR,QPCT,QPRT, QSOX1, RAB10, RAB11B, RAB17, RABA, RAB1B, RAB22A, RAB25, RAB29, RAB2A, RAB34, RAB3B, RAB3GAP1, RAB43, RAB4B, RAB6B, RABA, RAB A,RAB8B,RAB9A, RAC, RACGAP1, RALA, RALB, RAP1A, RAPB, RAP1GDS1, RAP2A, RAPGEF3, RARRES1, RARS, RASAL3, RASSF9, RBL2, RCC2, REG4, RELN, RENBP, RFC1, RFTN1, RHEB, RHOB, RHOF, RIMS2, RLF, RNASE7, RNF213, RNH1, RNPEP,ROBO2,ROCK2,RP2,RPL10,RPL10A,RPL14,RPL15, 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, RRAS, RREB1, RSU1, RTN4, RUSC2, RUVBL1, RUVBL2, RYR1, S100A11, S100A6, S100P, SAA1, SAFB2, SAMM50, SARS, SBSN, SCAMP2, SCAMP3, SCARB1, SCARB2, SCEL, SCIN, SCN10A, SCN11A, SCPEP1, SCRIB, SCRN2, SDCBP, SDF4, SEC31A, SELENBP1, SELP, SEMA3G, SEPP1, SERBP1, SERINC1, SERINC2, SERINC5, SERPINA1, SERPINA3, SERPINA4, SERPINA5, SERPINA7, SERPINB1, SERPINB13, SERPINB6, SERPINB9, SERPING1, SETD4, SFI1, SFN, SFRP1, SFT2D2, SH3BP4, SHMT1, SHMT2, SHROOM2, SIAE, SIRPA, SIT1, SLAMF1, SLAMF6, SLC12A1, SLC12A2, SLC12A3, SLC12A7, SLC12A9, SLC13A2, SLC13A3, SLC15A2, SLCA, SLC1A1, SLC1A4, SLC1A5, SLC20A2, SLC22A11, SLC22A12, SLC22A13, SLC22A2, SLC22A5, SLC22A6, SLC23A1, SLC25A1, SLC25A3, SLC25A4, SLC25A6, SLC26A11, SLC26A4, SLC26A9, SLC27A2, SLC29A1, SLC2A1, SLC2A3, SLC34A2, SLC35D1, SLC36A2, SLC37A2, SLC38A1, SLC39A5, SLC3A1, SLCA,SLC44A1, SLC44A2, SLC44A4, SLC46A3, SLC4A1, SLC4A4, SLC5A1, SLC5A10, SLC5A2, SLC5A5, SLC5A6, SLC5A8, SLC5A9, SLC6A13, SLC6A14, SLC6A19, SLC7A5, SLC9A1, SLC9A3, SLC9A3R1, SLC9A3R2, SLCO4C1, SLIT2, SLK, SMC2, SMC3, SMIM22, SMIM24, SMO, SMPDL3B, SMURF1, SNCG, SND1, SNRNP200, SNX12, SNX18, SNX25, SNX33, SNX9, SOD1, SOGA1, SORD, SORL1, SORT1, SPAG9, SPAST, SPEN, SPINK1, SPON2, SPRR3, SPTAN1, SPTBN1, SQSTM1, SRC, SRPR, SRSF7, ST13, ST3GAL1, ST3GAL6, STAMBP, STAU1, STIP1, STK10, STK11, STK24, STOM, STRIP1, STX3, STX4, STX7, STXBP1, STXBP2, STXBP3, STXBP4, SUB1, SUCLA2, SUSD2, SYAP1, SYNE1, SYNE2, TAB3, TACSTD2, TAF6L, TALDO1, TAOK1, TARS, TAX1BP1, TAX1BP3, TBC1D10A, TBC1D21, TC2N, TCP, TECTA, TEKT3, TEX14, TF, TFRC, TGFB1, TGFBI, TGFBR3, TGM1, TGM2, TGM3, TGM4, THBS, THBS2, THRAP3, THSD4, THY1, TIAM2, TINAGL1, TJP2, TKT, TLN1, TLR2, TM7SF3, TM9SF2, TMBIM1, TMC6, TMC8, TMED2, TMED9, TMEM109, TMEM192, TMEM2, TMEM256, TMEM27, TMEM63A, TMPRSS11B, TMPRSS11D, TMPRSS2, TNFAIP3, TNFRSF8, TNFSF10, TNFSF13, TNIK, TNKS1BP1, TNPO3, TOLLIP, TOM1, TOM1L2, TOMM70A, TOR1A, TOR1B, TOR3A, TPI, TPM3, TPP1, TPRG1L, TRAP1, TREH, TRIP10, TSNAXIP1, TSPAN1, TSPAN15, TSPAN3, TSSK3, TSTA3, TTC17, TTC18, TTLL3, TTN, TTR, TUBAB, TUBA4A, TUBB3, TUBB4A, TUBB8, TUFM, TWF2, TXNDC16, TXNDC8, TXNRD1, TYK2, TYRP1, UACA, UBA, UBAC1, UBASH3A, UBE2N, UBE2V2, UBL3, UBXN6, UEVLD, UGDH, UGGT1, UGP2, ULK3, UMOD, UPB1, UPK1A, UPK3A, UQCRC2, UTRN, UXS1, VAMP1, VAMP3, VAMP7, VAPA, VASN, VASP, VAT1, VCL, VCP, VDAC3, VIL1, VIM, VMO1, VPS13C, VPS13D, VPS28, VPS35, VPS36, VPS37B, VPS37C, VPS37D, VPS4A, VPS4B, VTA1, VWA2, VWF, WARS, WAS, WASF2, WASL, WDR1, WIZ, WNT5B, XDH, XPNPEP2, XPO1, 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]. Advances in Virology 7 훼-HIV Nef 250 kD 훼-HIV antigens Patient ID 27 28 30 41 108 104 103 86 48 Nef c1 c2 c3 p24 MW kD Figure 1: Detection of HIV- 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. (a) (b) 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.EVsfromHIV-1posi. Table 5: Exosomal proteins found in urinary EVs from uninfected controls. Genes in our Genes in the FunRich Fold enrichment Percentage of genes analysis database Exosomal proteins 37 2001 72.54 5.26 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. 8 Advances in Virology Table 6: Functional enrichment analysis of HIV+ EV proteins. Genes in the Genes in the Percentage of Corrected𝑃 Fold enrichment dataset Bkg. database genes value (BH FDR) Molecular function Protein serine/threonine kinase −08 272 5,602 30 1.18 1.04 activity −05 Catalytic activity 456 827 4.9 1.1 1.12 −05 GTPase activator activity 131 836 4.7 1.2 8.14 Guanyl-nucleoside exchange −05 105 614 3.6 1.2 8.54 factor activity Cell adhesion molecule activity 307 531 3.3 1.1 0.0001 Biological process Regulation of nucleobase, −05 2,236 4,658 24.8 1.05 3.24 nucleoside, and nucleic acid Biological pathway Integrin cell surface interactions 69 1,366 23.3 1.2 0.03 Table 7: Comparison of pathways between HIV+ groups from Pathway Studio 11.4. HIV group Pathway Natural killer cell inhibitor receptor signaling Intermediate filament polymerization Ca2+ u fl x regulation G1/S phase transition CD4+ T cells greater than 300 G2/M phase transition 𝑛 =15 S/G2 phase transition Protein folding Golgi to endosome transport Endosomal recycling Kinetochore assembly Neutrophil chemotaxis Vascular motility Platelet activation via GPCR signaling Insulin influence on protein synthesis mTOR signaling overview CD4+ T cells less than 300 EDNRA/B→ vascular motility 𝑛 =15 Proplatelet maturation Natural killer cell activation through ITAM-containing receptors Taste sensor receptors activates mTOR signaling Natural killer cell activation Intermediate filament polymerization Natural killer cell inhibitory receptor signaling golgi to endosome transport Ca+ u fl x regulation Low VLs HRH1/3→ synaptic transmission 𝑛 =14 Vascular motility Endosomal recycling G1/S phase transition Golgi transport G2/M phase transition Metaphase/anaphase phase transition S/G2 phase transition Spindle assembly High VLs Natural killer cell activation 𝑛 =10 Histone ubiquitylation Eosinophil survival by cytokine signaling Protein folding G2/M phase transition Advances in Virology 9 Max intensity (AU): 23 0 100 200 300 400 500 600 700 800 900 0 100 200 300 400 500 600 700 (nm) Particle size/Concentration Particle size/Relative intensity (a) Max intensity (AU): 22118 0 100 200 300 400 500 600 700 0 100 200 300 400 500 600 700 800 900 Particle size/concentration (nm) Particle size/relative intensity (b) Figure 3: Nanosight analysis (representative analysis). (a) NTA analysis of an HIV-negative urine sample had 0.4× 10 particles per ml (left panel) while (b) depicts an urine sample from a HIV+ patient that had 8.7× 10 particles per ml and has a greater relative intensity profile (right panel (a) and (b)) when compared to the HIV-negative sample. eTh Rank Sum 𝑇 test showed that HIV+ patient urine samples had more particles per ml than the negative control urine (𝑃<0.05). were also found by LC/MS/MS in addition to MHC Class I processes were immune response, signal transduction, cell and II antigens. communication, and antigen presentation (𝑃<0.0073). The HIV-1 Human Interaction database search found that Only sixty-four (64) proteins overlapped between the HIV Nef interacted with 559 EV proteins of 770 total human HIV+ and control EV samples and are listed in Table 8. proteins (72.6%); HIV Vpr interacted with 437 EV of 598 The top fourteen (14) GO ontologies for cellular components human (73.1%); HIV Vif interacted with 162 EV of 310 human include extracellular exosome, extracellular region, extracel- (52.2%); and HIV Vpu interacted with 165 EV of 244 human lular space, hemoglobin complex, and blood microparticle proteins which were found in the HIV+ EVs (67.6%) (see (𝑃 < 0.001, Table 9), GO ontologies for molecular function Supplementary Material 2, including PMIDs for references). were heparinbinding,iongatedactivity, andoxygentrans- Functional analysis of the control EVs are listed in Table 8. porter activity, and the most significant biological processes The major sites of expression were cervicovaginal uid fl , found were response to yeast, defense response to fungus, neutrophils, and gastric juice (𝑃<0.0001). The most signif- macrophage chemotaxis, negative regulation of growth of icant ontologies were molecular function of the proteins and symbiont in host, oxygen transport, and hydrogen peroxide defense/immunity protein activity and principal biological catabolic process. Con/ml E6 Con/ml E6 Int Int 10 Advances in Virology Cytoplasm 32.1 ∗∗ 75.4 ∗∗ Endothelium Plasma 67.9 ∗∗ Nucleus 32.4 ∗∗ Liver 65.5 ∗∗ Plasma mem ∗∗ 19.3 Kidney ∗∗ 57.8 Exosome 11.2 ∗∗ Lung 56.3 ∗∗ Lysosome 8.9 ∗∗ ∗∗ Serum 44.1 0 5 10 15 20 25 30 35 0 20406080 Percentage of proteins Percentage of proteins 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. Table 8: Functional enrichment analysis of control EV proteins. Genes in the Genes in the Bkg. Percentage of Corrected𝑃 value Fold enrichment database database genes (BH FDR ) Site of expression Cervicovaginal uid fl 16 544 12.0 4.2 2.59𝐸−06 Neutrophils 13 392 9.7 4.8 6.68𝐸−06 Gastric juice 9 222 6.7 6.1 4𝐸−05 Molecular function Defense/immunity protein activity 5 52 3.7 15.7 3.96𝐸−05 Biological process Immune response 13 561 9.8 3.4 0.00026 Signal transduction 43 3907 32.5 1.5 0.0026 Cell communication 41 3687 31.1 1.5 0.0028 Antigen presentation 1 1 0.7 134.4 0.0073 4. Discussion HIV+,1,932,and HIV−,only37.TEManalysisofHIV+and HIV− urine showed pleiotropic membrane bound vesicles in This is the rfi st report of the detection of urinary EVs both groups’ urine samples and NTA analysis showed parti- containing HIV and human proteins from HIV+ patients by cles ranging in size from 50 nm to 300 nm in both groups, mass spectrometry and western blot. EVs provide intercel- although the HIV+ samples had signicfi antly more particles lular communication to cells through the delivery of their than uninfected samples. Other studies have found increased cargo, nucleic acids, miRNAs, and proteins, to recipient cells numbers of EVs in the plasma of HIV+ patients [43, 49]. reviewed in [3]. Previous studies have found EVs in plasma of Proteins from both the HIV+ and HIV− individuals were sig- HIV+ patients but did not describe HIV or human proteins nicfi antlyassociatedwithexosomalproteins,furthersubstan- within them. Others have described EVs containing HIV tiating our hypothesis that urine from HIV+ patients contains proteins but these results were from in vitro HIV infected cell EVs (Table 10). eTh FunRich analysis of the sites of expression cultures and not from HIV+ patients [18, 20, 22, 23, 36, 38– showed that asignicfi antnumberofproteinswereassociated 47]. This study details both the HIV and human proteins with the endothelium, plasma, serum, kidney, liver, and lung. found in urinary EVs from HIV+ patients. These findings suggest that EVs from HIV+ patients may be According to the International Society for Extracellular filtered from these sites and concentrated in urine. Vesicles (ISEV), the minimal requirements for EVs or their HIV has previously been detected in the urine of HIV+ presence in samples includes the simultaneous detection of patients; however, it was shown that HIV virions are associ- transmembrane proteins and cytosolic proteins with mem- ated with cell pellets and not in centrifuged urine [50, 51]. brane/receptor binding abilities, while major cell organelles p24 is found in replicative HIV infectious virions but was are absent [48]. LC/MS/MS analysis identified these proteins not found in twenty-six of our HIV+ samples by ELISA and functional enrichment analysis determined a significant and only five of thirty-five HIV+ EV urine samples had number which were of exosomal origin in both the EVs in detectable p24 by LC/MS/MS analysis. p24 in urine pellets Site of expression Cellular component Advances in Virology 11 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 12 Advances in Virology Table 9: Continued. Gene 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 Bkg. Percentage of Corrected𝑃 value Fold enrichment data set database genes (Bonferroni method) Site of expression Urine 31 3202 51.7 3.0 6.85𝐸−07 Cervicovaginal uid fl 12 544 20.0 7.2 5.33𝐸−05 Neutrophils 9 392 15.0 7.7 1.56𝐸−03 032403 BALF4 glypep 4 43 6.7 35.0 3.53𝐸−03 Neutrophil 19 1979 31.7 3.0 3.67𝐸−03 Monocyte 23 2786 38.3 2.6 3.90𝐸−03 Cellular component Extracellular 22 1808 37.9 3.1 4.61𝐸−05 Stored secretory granule 3 19 5.2 51.0 3.27𝐸−03 Lysosome 17 1609 29.3 2.8 7.73𝐸−03 Extracellular space 8 399 13.8 5.6 1.05𝐸−02 Exosomes 19 2001 32.8 2.5 1.14𝐸−02 Azurophil granule 2 6 3.4 108.9 1.35𝐸−02 is derived from mononuclear cells but was found in only 3 203 and 311 days, after the rst fi sample that had similar results. of 80 analyzed samples [51]. This represents a low sensitivity, The identification of HIV proteins in urinary EVs may be a primarily because the HIV-1 p24 protein is not always present potential noninvasive diagnostic tool to monitor HIV disease during advanced stages of HIV infection. To further confirm states as well as treatment efficacy. that these HIV proteins were from EVs, we tested the filtrate Different proteins and pathways were found in EVs from from ultracentrifugation (MW cutoff 100,000 kD) of HIV- (1)CD4+Tcell> 300 versus<300 and (2) VLs< 200 versus positive urine, and no HIV proteins were present. We did >200 copies. It is interesting that EVs from HIV+ patients not, however, perform an HIV infectivity assay, MAGI, on the withlowVLsandhighCD4+Tcells,usuallyindicativeof isolated urinary EVs, and thus cannot be totally cond fi ent that better health, had more proteins detected than EVs from HIV virions were not present in the EVs. HIV proteins in uri- highVLsandlowCD4+Tcells(highVLs=2486vrslow nary EVs may be the result of a nonproductive HIV infection = 15028; low CD4+ T cells = 2115 versus high CD4+ = in the kidney [52–56] and/or EVs filtered from blood [21, 49, 15761). es Th e groups also had overlapping pathway results; 57]. The type of HIV protein in the EVs remained relatively however, proteins from high VLs and low CD4+ T cells did constant as demonstrated by the resampling of two patients, not have similar pathway results. Further comparison and Advances in Virology 13 analysis of the EV protein profile between the low VL/high One of the limitations of this study was a small sample CD4+ T cells and high VL/low CD4+ T cells may reveal size of specific HIV syndromes such as comorbidities, AIDS, more mechanisms involved in the evolving pathology of HIV HIV-associated nephropathy, and HIV-associated dementia infection. as well as patients on or na¨ıve to antiretroviral therapy. Proteins contained in EVs can both enhance and inhibit Increasing the numbers of HIV+ patients in these categories host responses from innate, inflammatory, and adaptive may allow us to determine whether specific HIV proteins as reactions. Proteins from HIV+ patients showed a predom- well as human proteins in urinary EVs could be associated inantly immunosuppressive profile. IL10 is a Th2 cytokine with these conditions. Future studies will also quantify the that downregulates macrophage function and inhibits T cell amount of HIV proteins as well as human proteins to deter- proliferation while IL6 can stimulate IL10 production and mine if a correlation exists between different HIV conditions inhibit the effects of TNF- 𝛼 and IL1. Both these cytokines and the amount of proteins detected. were present in the EVs from HIV+ patients while TNF-𝛼 HIV infection is usually detected by antibodies to HIV and IL1 were not detected suggesting an immunomodulatory and can take up to three months to develop or by measuring eect ff may be elicited by the EVs. Other immune downregu- VLs in blood whereas we can detect HIV-1 proteins in lating factors, IDO, HOXB4, HGF, and TGF𝛽1, were found. urinary EVs. In summary, urinary proteins in EVs from IDO [58], HLA-G [59], and HGF [60] can inhibit natural HIV+ patients may allow a noninvasive method to (1) rapidly killer cell activation which was one of the top biological screen forinfection andidenticfi ationofpatientseligiblefor processes found in the pathway analysis of the EV proteins antiretroviral treatment (ART); (2) monitor ART treatment in patients with high CD4+ T cells and low VLs. TGF𝛽-1, an efficacy; and (3) diagnose HIV comorbidities. inhibitor of immune function, is induced by HIV Tat [61] and isamediatorofimmunesuppressionin HIVinfection [62– Conflicts of Interest 64]. These proteins were found in EVs from HIV+ patients while proinflammatory cytokines were not. New studies show eTh authors declare that they have no conflicts of interest. that HIV+ nonprogressors have lower plasma TGF𝛽-1 and IL10 than patients with progressive disease [65] and it is Acknowledgments possible that EVs may sequester TGF𝛽-1 and IL10 and remove them from circulation. The presence of over 16 different The authors acknowledge Jane Chu and Mahfuz Khan of MHC Class I and II antigens in the EVs from HIV+ patients Morehouse School of Medicine for technical assistance and may support the hypothesis that this mechanism is used Dr. Douglas Paulsen for his support and editorial sugges- by intracellular pathogens to evade the immune response tions. This study was supported by the National Center for by decreasing cytotoxic T cell activity [66]. Herpes Simplex Advancing Translational Sciences of the National Institutes Virus-1 binds to HLA-DR inhibiting antigen presentation that of Health under Award no. UL1TR000454. Other funds were leads to immune evasion [67]. Future studies should focus on received from the Minority Biomedical Research Support the correlation of the concentration of these factors to HIV+ (MBRS) of the Research Initiative for Scientific Advancement patients’ clinical status. (RISE) Program 5R25GM058268 funded by NIGMS and In this study, we showed that structural, regulatory, and NIH Research Endowment S21MD000101 funded by the accessory HIV proteins could be detected in urinary EVs of National Institute on Minority Health and Health Disparities HIV+ patients. Our WB analysis using polyclonal and mon- (NIH/NIMHD). The authors also acknowledge the Research oclonal antibodies conrfi med the presence of HIV proteins Centers in Minority Institutions (RCMI) G12 funded by in the EVs from HIV+ patients. eTh most prevalent protein the NIH/NIMHD, #8G12MD0076202. MEB Core facility wasHIV Nef. EVsfromboth in vitro and patient samples was constructed with support from the Research Facili- have been previously reviewed in [6]. HIV Nef induces an ties Improvement Grant C06 RR18386 from NIH/NCRR. alternative pathway for TNF induction utilizing Notch-1, The newly renovated Core Resources space was funded ADAM17, and Rab4+, all found in EVs from HIV+ patients, by G20 RR031196 from NIH/NCRR. The R-CENTER was which leads to high plasma TNF levels [68]. Whether the funded by Grant no. U54MD007588 from NIH/NIMHD and isolation of these factors in EVs represents a diminishing or NIH/NCRR 5P20R R0111044 pilot for study support. enhancement of TNF production remains to be examined. The HIV Human Interaction database found significant Supplementary Materials interactions between HIV Nef, Vpr, Vif, and Vpu and human proteins. Serine/threonine protein kinases are important in Supplementary . Top 10 biological function pathways using T cell receptor signaling [69]. es Th e kinases as well as CD4 Pathway Studio 11 Mammal Plus, Elsevier, Inc., for HIV+ EV and MHC antigens were found in EVs from the HIV+ proteins from HIV+ patients with (1) CD4+ T cells greater samples; however, further studies are needed to determine the than 300, (2) CD4+ T cells less than 300, (3) viral loads less mechanisms involved with EV function in HIV infections. than 200 copies, and (4) viral loads greater than 200 copies. Cell adhesion molecules, ICAM, VCAM, and PECAM, were also found in the EVs from patients. Others have reported Supplementary . 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