the u937/nef - eyfp and u937/eyfp cells and their culture conditions have been described previously ( 17 ) . exosomes were prepared from culture supernatants ( 18 ) and visualized by electron microscopy as described earlier ( 17 ) . exosome quantification was done using bradford reagent ( biorad , usa ) as described elsewhere ( 18 ) . for flow cytometry , purified exosomes ( ~35 g protein ) were first incubated with 10 l of aldehyde latex beads for 15 minutes at room temperature . then pbs was added to a final volume of 1 ml and incubated on a rotator for 2 hour at room temperature , or overnight at 4c . to this 110 l of 1 m glycine ( final concentration , 100 mm ) the beads were then centrifuged for 3 minutes at 4,000 rpm at room temperature and washed twice with 1 ml pbs/0.5% bsa with final re - suspension in 0.5 ml pbs - bsa . for staining with antibodies , 10 l of coated beads were incubated with 50 l of the appropriate antibody diluted in pbs - bsa for 30 minutes at 4c . the beads were then washed twice as described above followed by incubation with 50 l secondary antibodies for 30 minutes at 4c . the beads were again washed twice and were acquired on a cyan - adp flow cytometer ( beckman coulter ) . data were analysed using the flow jo software . for the labelling of exosomal membranes with pkh26 , a fluorescent lipid dye ( sigma ) the excess dye was removed by washing exosomes with 3 ml of pbs with ultracentrifugation at 100,000g in a sw60 rotor at 4c . the labelled exosomes were also bound to aldehyde latex beads as described above and beads were observed under 60 magnification in the confocal microscope . total rna was isolated from cells or exosomes using trizol ( invitrogen ) as per manufacturer 's instructions . approximately 10 million cells or 50100 g exosomes were re - suspended in 500 l of trizol and vortexed thoroughly . this was left for 10 minutes at room temperature and then centrifuged at 13,000 rpm at 4c for 10 minutes . the upper aqueous phase was mixed with 2 volumes of isopropanol and incubated overnight at 20c . the pellet was washed with 70% ethanol , air - dried and dissolved in depc - treated water . rna preparations were treated with dnasei ( new england biolabs , usa ) according to manufacturer 's instructions before further use . this rna was used for messenger rna analysis and for the taqman mirna array profiling . the mirnas were isolated using the mirneasy kit ( qiagen , germany ) following the supplier 's protocol . briefly , ~23 million cells or 80100 g exosomes were harvested and lysed in ~750 l qiazol . the mixture was vortexed for a few seconds and centrifuged at 13,000 rpm for 10 minutes at 4c . an equal amount of ethanol was added to the supernatant and loaded on the columns , which were washed with the buffers provided in the kit , and eluted in ~50 l nuclease - free water . the rna amount and quality were estimated by absorbance values at 260 and 280 nm on a nanodrop spectrophotometer . total rna was isolated as above using trizol and the mirna profiling was carried out at labindia ( gurgaon , india ) using the applied biosystems taqman low density array human microrna array version 2.0 , which covered 667 known human micrornas , and included 6 endogenous mirna controls the small rna fraction was purified from total rna pool using the mirvana mirna isolation kit ( qiagen ) , which was converted to cdnas using megaplex rt primers and taqman microrna reverse transcription kit . a pre - amplification step was carried out using megaplex preamp primers and taqman preamp master mix . the cdna was diluted , taqman universal pcr master mix was added and the mixture was loaded on a 384-well format taqman array . data were analysed using data assist software v3.0.1 with the threshold ct value as 35 . for all analyses , the levels in control samples were set as 1 , and the levels in the test sample were calculated with the software . one g total rna from u937/nef - eyfp and u937/eyfp cells , or 50100 ng from exosomes was reverse transcribed with a specific stem - loop ( sl ) primer ( supplementary file ) . the reaction mix was prepared as described above and incubated as follows : 70c for 5 minutes followed by 7 cycles of 16c for 5 minutes and 42c for 5 minutes . after heating at 85c for 5 minutes , the mixture was either used immediately for quantitative pcr or stored at 70c . an appropriate amount of cdna was first used to optimize the rnu6b signal in each sample . subsequently , amplifications of specific mature mirnas were carried out using the primers shown in supplementary file . for the quantification of mrnas , total rna was converted to cdna using a kit ( promega , usa ) according to the manufacturer 's protocol . briefly , a reaction mix was prepared using 1 g total rna , 500 ng oligo(dt)20 and depc water . the reaction mixture was incubated at 65c for 5 minutes and chilled on ice for at least 1 minute . the following components were then added in the given order : 10 l of 5x promega rt buffer , 0.25 l rnasein ( recombinant rnase inhibitor ; 40 u/l ) , 0.5 l of mulv rt enzyme ( 400 u/l ) and 0.5 l of dntps ( 10 mm ) . the reaction was incubated at 42c for 60 minutes , followed by heating at 70c for 15 minutes and then chilled on ice . a 20-l reaction mix was prepared using 2x evagreen dye , 12 l of cdna , and 510 pmoles each of forward and reverse primers . the amplification conditions were as follows : 94c for 10 minutes , followed by 40 cycles of 94c for 30 seconds , 6062c for 30 seconds , 72c for 30 seconds , and a final extension at 72c for 2 minutes . the ct values of replicates were analysed to calculate relative fold change by the ct method . the mirwalk database ( http://www.umm.uniheidelberg.de/apps/zmf/mirwalk/ ) ( 19 ) was searched to identify validated target genes of the selected mirnas , followed by pathway analysis using the david bioinformatics database ( http://david.abcc.ncifcrf.gov/gene2gene.jsp ) ( 20 , 21 ) . microinspector ( http://bioinfo.uni-plovdiv.bg/microinspector/ ) ( 22 ) and rna hybrid ( http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/ ) ( 23 ) algorithms were used to identify mirnas targeting hiv-1 strains indie - c1 ( accession no . all of the 2,578 mature human mirnas available in mirbase version 20 were analysed using a free - energy cut - off of 14 kcal / mole . we also compared the list of validated targets of mirnas upregulated or downregulated in u937/nef - eyfp cells with genes that are reported in literature to be upregulated or downregulated by nef ; the latter information is part of the hiv , human protein interaction database and is available at http://www.ncbi.nlm.nih.gov/projects/refseq/hivinteractions/nef.html . cells were washed twice in cold 1x pbs ( 137 mm nacl , 2.7 mm kcl , 10 mm na2hpo4 , 18 mm kh2po4 ) and lysed in 1x ripa buffer ( 20 mm tris - hcl ( ph 7.5 ) , 150 mm nacl , 1 mm na2edta , 1 mm egta , 1% np-40 , 1% sodium deoxycholate , 2.5 mm sodium pyrophosphate , 1 mm -glycerophosphate , 1 mm na3vo4 , 1 g / ml leupeptin ) . cell lysis was carried out for 45 minutes at 4c with gentle vortexing every 10 minutes . the protein concentration of the lysate was estimated using the bradford reagent ( bio - rad , usa ) . for western blotting , 100 microgram of cell lysate was boiled in laemmli buffer , and the proteins were separated by sodium dodecyl sulphate - polyacrylamide gel electrophoresis ( sds - page ) . the separated proteins were transferred to a nitrocellulose membrane ( hybond ecl ; amersham biosciences ) at constant voltage of 65 v for 60 minutes . the membrane was then blocked with tris - buffered saline ( tbs ) containing 3% blotto ( bio - rad ) for 1 hour at room temperature and washed with tbst ( tbs containing 0.1% tween 20 ) . the membrane was then incubated overnight at 4c with the primary antibody appropriately diluted in tbst3% blotto , washed 4 times for 10 minutes each with tbst , and incubated with horseradish peroxidase - linked secondary antibodies diluted in tbst5% blotto for 1 hour at room temperature . after the membrane was washed as described above , chemiluminescent detection of proteins was carried out using luminol reagent ( santa cruz biotechnology , usa ) according to the supplier 's protocol . primary antibodies anti - il6 ( sc7920 ) , anti - il1 ( sc1250 ) , anti - actin and hrp - linked secondary antibodies were from santa cruz biotechnology ( usa ) . the u937/nef - eyfp and u937/eyfp cells and their culture conditions have been described previously ( 17 ) . exosomes were prepared from culture supernatants ( 18 ) and visualized by electron microscopy as described earlier ( 17 ) . exosome quantification was done using bradford reagent ( biorad , usa ) as described elsewhere ( 18 ) . for flow cytometry , purified exosomes ( ~35 g protein ) were first incubated with 10 l of aldehyde latex beads for 15 minutes at room temperature . then pbs was added to a final volume of 1 ml and incubated on a rotator for 2 hour at room temperature , or overnight at 4c . to this 110 l of 1 m glycine ( final concentration , 100 mm ) the beads were then centrifuged for 3 minutes at 4,000 rpm at room temperature and washed twice with 1 ml pbs/0.5% bsa with final re - suspension in 0.5 ml pbs - bsa . for staining with antibodies , 10 l of coated beads were incubated with 50 l of the appropriate antibody diluted in pbs - bsa for 30 minutes at 4c . the beads were then washed twice as described above followed by incubation with 50 l secondary antibodies for 30 minutes at 4c . the beads were again washed twice and were acquired on a cyan - adp flow cytometer ( beckman coulter ) . data were analysed using the flow jo software . for the labelling of exosomal membranes with pkh26 , a fluorescent lipid dye ( sigma ) the excess dye was removed by washing exosomes with 3 ml of pbs with ultracentrifugation at 100,000g in a sw60 rotor at 4c . the labelled exosomes were also bound to aldehyde latex beads as described above and beads were observed under 60 magnification in the confocal microscope . total rna was isolated from cells or exosomes using trizol ( invitrogen ) as per manufacturer 's instructions . approximately 10 million cells or 50100 g exosomes were re - suspended in 500 l of trizol and vortexed thoroughly . this was left for 10 minutes at room temperature and then centrifuged at 13,000 rpm at 4c for 10 minutes . the upper aqueous phase was mixed with 2 volumes of isopropanol and incubated overnight at 20c . the pellet was washed with 70% ethanol , air - dried and dissolved in depc - treated water . rna preparations were treated with dnasei ( new england biolabs , usa ) according to manufacturer 's instructions before further use . this rna was used for messenger rna analysis and for the taqman mirna array profiling . the mirnas were isolated using the mirneasy kit ( qiagen , germany ) following the supplier 's protocol . briefly , ~23 million cells or 80100 g exosomes were harvested and lysed in ~750 l qiazol . the mixture was vortexed for a few seconds and centrifuged at 13,000 rpm for 10 minutes at 4c . an equal amount of ethanol was added to the supernatant and loaded on the columns , which were washed with the buffers provided in the kit , and eluted in ~50 l nuclease - free water . the rna amount and quality were estimated by absorbance values at 260 and 280 nm on a nanodrop spectrophotometer . total rna was isolated as above using trizol and the mirna profiling was carried out at labindia ( gurgaon , india ) using the applied biosystems taqman low density array human microrna array version 2.0 , which covered 667 known human micrornas , and included 6 endogenous mirna controls the small rna fraction was purified from total rna pool using the mirvana mirna isolation kit ( qiagen ) , which was converted to cdnas using megaplex rt primers and taqman microrna reverse transcription kit . a pre - amplification step was carried out using megaplex preamp primers and taqman preamp master mix . the cdna was diluted , taqman universal pcr master mix was added and the mixture was loaded on a 384-well format taqman array . data were analysed using data assist software v3.0.1 with the threshold ct value as 35 . for all analyses , the levels in control samples were set as 1 , and the levels in the test sample were calculated with the software . one g total rna from u937/nef - eyfp and u937/eyfp cells , or 50100 ng from exosomes was reverse transcribed with a specific stem - loop ( sl ) primer ( supplementary file ) . the reaction mix was prepared as described above and incubated as follows : 70c for 5 minutes followed by 7 cycles of 16c for 5 minutes and 42c for 5 minutes . after heating at 85c for 5 minutes , the mixture was either used immediately for quantitative pcr or stored at 70c . an appropriate amount of cdna was first used to optimize the rnu6b signal in each sample . subsequently , amplifications of specific mature mirnas were carried out using the primers shown in supplementary file . for the quantification of mrnas , total rna was converted to cdna using a kit ( promega , usa ) according to the manufacturer 's protocol . briefly , a reaction mix was prepared using 1 g total rna , 500 ng oligo(dt)20 and depc water . the reaction mixture was incubated at 65c for 5 minutes and chilled on ice for at least 1 minute . the following components were then added in the given order : 10 l of 5x promega rt buffer , 0.25 l rnasein ( recombinant rnase inhibitor ; 40 u/l ) , 0.5 l of mulv rt enzyme ( 400 u/l ) and 0.5 l of dntps ( 10 mm ) . the reaction was incubated at 42c for 60 minutes , followed by heating at 70c for 15 minutes and then chilled on ice . a 20-l reaction mix was prepared using 2x evagreen dye , 12 l of cdna , and 510 pmoles each of forward and reverse primers . the amplification conditions were as follows : 94c for 10 minutes , followed by 40 cycles of 94c for 30 seconds , 6062c for 30 seconds , 72c for 30 seconds , and a final extension at 72c for 2 minutes . the ct values of replicates were analysed to calculate relative fold change by the ct method . the mirwalk database ( http://www.umm.uniheidelberg.de/apps/zmf/mirwalk/ ) ( 19 ) was searched to identify validated target genes of the selected mirnas , followed by pathway analysis using the david bioinformatics database ( http://david.abcc.ncifcrf.gov/gene2gene.jsp ) ( 20 , 21 ) . microinspector ( http://bioinfo.uni-plovdiv.bg/microinspector/ ) ( 22 ) and rna hybrid ( http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/ ) ( 23 ) algorithms were used to identify mirnas targeting hiv-1 strains indie - c1 ( accession no . all of the 2,578 mature human mirnas available in mirbase version 20 were analysed using a free - energy cut - off of 14 kcal / mole . we also compared the list of validated targets of mirnas upregulated or downregulated in u937/nef - eyfp cells with genes that are reported in literature to be upregulated or downregulated by nef ; the latter information is part of the hiv , human protein interaction database and is available at http://www.ncbi.nlm.nih.gov/projects/refseq/hivinteractions/nef.html . cells were washed twice in cold 1x pbs ( 137 mm nacl , 2.7 mm kcl , 10 mm na2hpo4 , 18 mm kh2po4 ) and lysed in 1x ripa buffer ( 20 mm tris - hcl ( ph 7.5 ) , 150 mm nacl , 1 mm na2edta , 1 mm egta , 1% np-40 , 1% sodium deoxycholate , 2.5 mm sodium pyrophosphate , 1 mm -glycerophosphate , 1 mm na3vo4 , 1 g / ml leupeptin ) . cell lysis was carried out for 45 minutes at 4c with gentle vortexing every 10 minutes . the protein concentration of the lysate was estimated using the bradford reagent ( bio - rad , usa ) . for western blotting , 100 microgram of cell lysate was boiled in laemmli buffer , and the proteins were separated by sodium dodecyl sulphate - polyacrylamide gel electrophoresis ( sds - page ) . the separated proteins were transferred to a nitrocellulose membrane ( hybond ecl ; amersham biosciences ) at constant voltage of 65 v for 60 minutes . the membrane was then blocked with tris - buffered saline ( tbs ) containing 3% blotto ( bio - rad ) for 1 hour at room temperature and washed with tbst ( tbs containing 0.1% tween 20 ) . the membrane was then incubated overnight at 4c with the primary antibody appropriately diluted in tbst3% blotto , washed 4 times for 10 minutes each with tbst , and incubated with horseradish peroxidase - linked secondary antibodies diluted in tbst5% blotto for 1 hour at room temperature . after the membrane was washed as described above , chemiluminescent detection of proteins was carried out using luminol reagent ( santa cruz biotechnology , usa ) according to the supplier 's protocol . primary antibodies anti - il6 ( sc7920 ) , anti - il1 ( sc1250 ) , anti - actin and hrp - linked secondary antibodies were from santa cruz biotechnology ( usa ) . exosomes isolated from u937/eyfp and u937/nef - eyfp cells using a differential centrifugation protocol were found by electron microscopy to be of the expected size of 3090 nm ( fig . these were also tested for the exosomal marker proteins alix , tsg101 and cd81 , which were present in purified exosomes as well as in both types of cells . however , unlike cell lysates we did not detect voltage - dependent anion channel ( vdac ) , calnexin or cytochrome c in the exosome preparation , indicating that it was not contaminated with mitochondria , endoplasmic reticulum or apoptotic bodies ( 17 ) . we further detected cd81 on the surface of exosomes prepared from both cell lines by flow cytometry ( fig . the exosomes were also labelled with pkh26 , a red fluorescent lipid dye , and were visualized by confocal microscopy either directly ( fig . 1c , panels a and b ) or after binding to aldehyde latex beads ( fig . we also quantified exosome secretion from the two cell lines by normalizing total exosomal protein with the number of cells harvested for exosome preparation . the average exosome yields were 0.384 g / million u937/eyfp cells and 0.615 g / million u937/nef - etfp cells ( fig . thus , the nef - expressing u937 cells secreted on an average about 60% more exosomes than control cells . ( a ) electron microscopy of exosomes from u937/nef - eyfp cells ; bar 100 nm . ( c ) exosomes were labelled with lipid dye pkh26 ; these were ( a , b ) observed directly at 100x magnification , or ( c , d ) first bound to 4 m latex beads and then observed at 100x magnification . ( d ) amount of exosomes secreted by u937/eyfp and u937/nef - eyfp cell lines presented as protein concentration from 110 cells . the rna isolated from both cell lines and their exosomes was analysed on an agilent bioanayzer . the yield for cellular rna was 1.177 g/l for u937/eyfp cells and 2.055 g/l for u937/nef - eyfp cells with rin of 9.4 and 9.1 , respectively ( supplementary file ) . the high rin values indicate the extracted rna to be of high quality . the yields for exosomal rnas were 53 and 52 ng/l for control and nef exosomes , respectively . the bioanalyzer profiles showed 2 distinct peaks of ribosomal rnas in the cellular rna pool , which is characteristic of the 18s and 28s species that were absent in the exosomal rna pool ( supplementary file ) . on the contrary , the exosomal rnas showed a major peak around 25 nt demonstrating the enrichment of small rnas in exosomes . the mirna population was then isolated from both cellular and exosomal rna pools using the mirvana mirna isolation kit and profiling was carried out using taqman low density arrays . the mirnas detected in u937/nef - eyfp cellular and exosomal pools were then compared to their respective control pools to determine differential cellular expression and exosomal secretion of mirnas in nef - expressing u937 monocytic cells . the results from cellular and exosomal datasets were then compared to identify the mirnas that were preferentially packaged in or excluded from nef - containing exosomes . in any sample , only ~50% of the 667 profiled mirnas were detected and the rest remained undetected for each pool . this showed that not all mirnas were produced in u937 cells , confirming the cell - specific expression of mirnas . the profiling data also suggested that cellular and exosomal mirna pools of u937/nef - eyfp cells vary considerably from that of control u937/eyfp cells . of the 667 mirnas analysed , 320 mirnas ( i.e. 48% ) were detected in u937/nef - eyfp and u937/eyfp cells , of which 87 ( 27% ) were expressed at 1.4-fold or higher levels ( mincup ) and 67 ( 21% ) were expressed at lower than 0.8-fold ( mincdown ) in u937/nef - eyfp compared to u937/eyfp cells ; the remaining 166 ( 52% ) mirnas were expressed at similar levels in both cell lines ( fig . analogous to the cellular pool , ~50% of the profiled mirnas were detected in any of the exosome samples and the rest remained undetected . of the 349 mirnas detected in nef exosomes , 311 ( 89% ) were present at 1.4-fold or higher levels ( mineup ) and 28 ( 8% ) were present at lower that 0.8 fold ( minedown ) in exosomes from u937/nef - eyfp compared to u937/eyfp cells ; the remaining 10 ( 3% ) were present at similar levels in exosomes from both cell lines ( fig . while the mirna profile of nef - expressing cells showed almost similar percentages of upregulated and downregulated mirnas , the exosomes from these cells contained increased levels of most of the detected mirnas . the mirnas in ( a ) cells and ( b ) exosomes were profiled and relative fold - changes calculated as described in methods . ( c ) the venn diagrams show the intersection of detected mirnas as follows : ( i ) upregulated mirnas in both u937/nef - eyfp cells and exosomes ; ( ii ) downregulated mirnas in u937/nef - eyfp cells and upregulated mirnas in u937/nef - eyfp exosomes ; ( iii ) upregulated mirnas in u937/nef - eyfp cells and downregulated mirnas in u937/nef - eyfp exosomes . we then examined mirnas upregulated in both cellular and exosomal pools of u937/nef - eyfp compared to u937/eyfp cells ( mincupmineup ) . there were 87 and 311 in nef - expressing cells and exosomes , respectively , of which 77 mirnas were common to these groups ( fig . these 77 mirnas are likely to be packaged more in nef exosomes because their levels are also higher in nef - expressing cells . to identify mirnas selectively secreted in nef exosomes ( minesel ) we compared the mirnas down regulated in nef - expressing cells with those that were upregulated in nef exosomes ( mincdownnmineup ) and found 47 mirnas in this category ( fig . 2cii , supplementary file ) . finally , we analysed mirnas that are selectively retained in nef - expressing cells ( mincsel ) and are not exported out through exosomes . for this , we compared the group of mirnas upregulated in nef - expressing cells with those that were downregulated in nef exosomes ( mincupminedown ) , and found only 2 such mirnas ( fig . thus , nef expression leads to the selective secretion of many more mirnas in exosomes compared to those that are selectively retained in nef - expressing cells . to confirm the profiling data , we selected 13 mirnas for validation by stem - loop quantitative rt - pcr in multiple replicates of cells and exosomes . these include mir-573 and mir-638 ( upregulated in nef cells ) , mir-548d-3p and 564 ( same levels in nef and control cells ) , and mir-16 - 1 , mir-146a , mir-125b , mir-146b-3p , mir-181a , mir-27a , mir-570 , mir-610 and mir-624 * ( down regulated in nef cells ) . all these mirnas , except mir-146a , were upregulated in exosomes in the profiling array data . as there is no well documented control small rna for exosomes , we quantified rnu6 levels in both nef and control exosomes , and found these to be almost similar in multiple replicates of the 2 populations . the average ct values obtained were 23.3 in control exosomes and 23.7 in nef exosomes ( supplementary file ) . the results showed most of the mirna levels to correlate with the profiling data . in the cells we found 4 mirnas to show a trend opposite to the profiling data . these include mir-27a , which is upregulated , and mir-548d-3p , mir-573 and mir-638 , which are down regulated in nef - expressing cells based on qrt - pcr analysis ( fig . all the analysed mirnas showed higher levels in nef exosomes , which is consistent with the profiling data ( fig . total rna isolated from ( a ) cells and ( b ) exosomes was used to quantify expression levels of selected mirnas as described in methods . since each mirna can potentially target hundreds of transcripts , changes in their abundance can have significant effects on cellular pathways . the validated targets of mirnas that were selectively secreted from nef - expressing cells were extracted using the mirwalk software . a majority of these mirnas were found to target the expression of cytokines / chemokines ( e.g. interleukins ) , proteins involved in innate immunity ( e.g. interferon , nfkb , stats ) , tumour suppressors ( pten , rb1 ) , cell survival factors ( bcl2 , mcl1 ) and the hiv restriction factor apobec3 g . in monocytes , nef induces the synthesis of proinflammatory cytokines and chemokines . this effect is observed with nef expressed intracellularly as well as that added exogenously to monocytes ( 24 ) . il1 , il1 , il6 , tnf , mip1 , mip1 , which are induced in the presence of nef , and observed that many of the mirnas that target these cytokines , such as mir-146a , mir-146b-3p , mir-125b and mir-181a , are downregulated in nef - expressing cells , but are selectively secreted in exosomes from these cells . these 4 mirnas regulate proinflammatory cytokine levels , antigen processing and presentation pathways ( 25 ) . we quantified the expression levels of il1 , il1 , il6 , tnf , mip1 , mip1 , and il10 mrnas in u937/nef - eyfp and u937/eyfp cells . except for tnf , which did not show any significant change , all other mrnas were present at much higher levels in nef - expressing cells ( fig . the results showed > 10-fold increase for il1 , il6 and mip1 mrnas , and about 5-fold increase for il10 and mip1 mrnas . for 2 of these , il1 and il6 , the protein expression levels were also estimated by western blotting ; these were also higher in nef - expressing cells ( fig . ( a ) rna isolated from u937/nef - eyfp and u937/eyfp cells were tested for the expression of selected cytokine genes by qrt - pcr ; relative fold - changes were calculated from raw ct values using the data assist software ( abi ) . error bars represent standard errors from 3 experiments , each with triplicate measurements ; * p<0.05 . ( b ) western blots of lysates from u937/nef - eyfp and u937/eyfp cells for il1 and il6 levels . host mirnas can also target hiv transcripts leading to a reduced level of viral replication . we then analysed our profiling data to identify the status of mirnas that can potentially target the hiv genome . in silico analysis was performed with the 451 detected mirnas that were matched against 2 hiv-1 genomes  nl4 - 3 ( clade b ) and indiec1 ( clade c ) , using two separate software tools  microinspector and rnahybrid . of the 262 potential hiv-1 targeting mirnas identified through this process , 189 were also present in exosomes from the 2 cell lines . interestingly , 170 of these mirnas were upregulated and only 19 were downregulated in exosomes from u937/nef - eyfp compared to u937/eyfp cells ( fig . this overwhelming preference for the secretion of potentially hiv - targeting mirnas suggests that nef - induced vesicle secretion might be a unique viral strategy to selectively export virus targeting host mirnas . ( a ) diagram showing the correlation between hiv targeting mirnas and nef exosomal mirnas . all the detected mirnas were screened for potential binding sites on 2 different hiv-1 genomes ( nl4 - 3 and indiec1 ) using the microinspector algorithm ( http://bioinfo.uni-plovdiv.bg/microinspector/ ) ; the identified mirnas were verified using the rnahybrid algorithm ( http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/ ) . ( b ) mirnas present in exosomes that can potentially target the hiv-1 genome are separated based on whether their levels are higher or lower in nef compared to control exosomes . using mirwalk , we obtained 1,565 validated target genes for the 47 mirnas that were selectively secreted in nef exosomes ( minesel ; supplementary file ) and 107 validated target genes of the 2 mirnas that were selectively retained in nef - expressing cells ( mincsel ; supplementary file ) . these were then subjected to pathway analysis using the david ( database for annotation , visualization and integrated discovery ) bioinformatics resources . we identified 63 kegg pathways ( p<0.05 ) corresponding to the 1,565 validated targets of the 47 mirnas in minesel . these include tgf - beta signalling , toll - like receptor signalling , apoptosis , cell cycle , t - cell receptor signalling , p53 signalling , mitogen activated protein kinase ( mapk ) signalling , cytokine  cytokine receptor interaction , janus kinase - signal transducer and activator of transcription ( jak - stat ) signalling , b - cell receptor signalling , mtor signalling and chemokine signalling ( supplementary file ) . we also identified 26 kegg pathways ( p<0.05 ) corresponding to the 107 validated targets of the mirnas in mincsel . some important pathways here include cell cycle , mapk signalling , p53 signalling , t - cell receptor signalling , toll - like receptor signalling , cytokine  cytokine receptor interaction and regulation of actin cytoskeleton ( supplementary file ) . we further compared the entries reported in the hiv , human protein interaction database to be upregulated and downregulated by nef with the mirna targets identified in this study . this identified 19 proteins ( genes ) reported to be upregulated by nef to also be present in our set of 1,565 upregulated genes ; similarly , 2 proteins ( genes ) were in common with our set of 107 downregulated genes . exosomes isolated from u937/eyfp and u937/nef - eyfp cells using a differential centrifugation protocol were found by electron microscopy to be of the expected size of 3090 nm ( fig . these were also tested for the exosomal marker proteins alix , tsg101 and cd81 , which were present in purified exosomes as well as in both types of cells . however , unlike cell lysates we did not detect voltage - dependent anion channel ( vdac ) , calnexin or cytochrome c in the exosome preparation , indicating that it was not contaminated with mitochondria , endoplasmic reticulum or apoptotic bodies ( 17 ) . we further detected cd81 on the surface of exosomes prepared from both cell lines by flow cytometry ( fig . the exosomes were also labelled with pkh26 , a red fluorescent lipid dye , and were visualized by confocal microscopy either directly ( fig . 1c , panels a and b ) or after binding to aldehyde latex beads ( fig . we also quantified exosome secretion from the two cell lines by normalizing total exosomal protein with the number of cells harvested for exosome preparation . the average exosome yields were 0.384 g / million u937/eyfp cells and 0.615 g / million u937/nef - etfp cells ( fig . thus , the nef - expressing u937 cells secreted on an average about 60% more exosomes than control cells . ( a ) electron microscopy of exosomes from u937/nef - eyfp cells ; bar 100 nm . ( c ) exosomes were labelled with lipid dye pkh26 ; these were ( a , b ) observed directly at 100x magnification , or ( c , d ) first bound to 4 m latex beads and then observed at 100x magnification . ( d ) amount of exosomes secreted by u937/eyfp and u937/nef - eyfp cell lines presented as protein concentration from 110 cells . the rna isolated from both cell lines and their exosomes was analysed on an agilent bioanayzer . the yield for cellular rna was 1.177 g/l for u937/eyfp cells and 2.055 g/l for u937/nef - eyfp cells with rin of 9.4 and 9.1 , respectively ( supplementary file ) . the high rin values indicate the extracted rna to be of high quality . the yields for exosomal rnas were 53 and 52 ng/l for control and nef exosomes , respectively . the bioanalyzer profiles showed 2 distinct peaks of ribosomal rnas in the cellular rna pool , which is characteristic of the 18s and 28s species that were absent in the exosomal rna pool ( supplementary file ) . on the contrary , the exosomal rnas showed a major peak around 25 nt demonstrating the enrichment of small rnas in exosomes . the mirna population was then isolated from both cellular and exosomal rna pools using the mirvana mirna isolation kit and profiling was carried out using taqman low density arrays . the mirnas detected in u937/nef - eyfp cellular and exosomal pools were then compared to their respective control pools to determine differential cellular expression and exosomal secretion of mirnas in nef - expressing u937 monocytic cells . the results from cellular and exosomal datasets were then compared to identify the mirnas that were preferentially packaged in or excluded from nef - containing exosomes . in any sample , only ~50% of the 667 profiled mirnas were detected and the rest remained undetected for each pool . this showed that not all mirnas were produced in u937 cells , confirming the cell - specific expression of mirnas . the profiling data also suggested that cellular and exosomal mirna pools of u937/nef - eyfp cells vary considerably from that of control u937/eyfp cells . of the 667 mirnas analysed , 320 mirnas ( i.e. 48% ) were detected in u937/nef - eyfp and u937/eyfp cells , of which 87 ( 27% ) were expressed at 1.4-fold or higher levels ( mincup ) and 67 ( 21% ) were expressed at lower than 0.8-fold ( mincdown ) in u937/nef - eyfp compared to u937/eyfp cells ; the remaining 166 ( 52% ) mirnas were expressed at similar levels in both cell lines ( fig . analogous to the cellular pool , ~50% of the profiled mirnas were detected in any of the exosome samples and the rest remained undetected . of the 349 mirnas detected in nef exosomes , 311 ( 89% ) were present at 1.4-fold or higher levels ( mineup ) and 28 ( 8% ) were present at lower that 0.8 fold ( minedown ) in exosomes from u937/nef - eyfp compared to u937/eyfp cells ; the remaining 10 ( 3% ) were present at similar levels in exosomes from both cell lines ( fig . while the mirna profile of nef - expressing cells showed almost similar percentages of upregulated and downregulated mirnas , the exosomes from these cells contained increased levels of most of the detected mirnas . the mirnas in ( a ) cells and ( b ) exosomes were profiled and relative fold - changes calculated as described in methods . ( c ) the venn diagrams show the intersection of detected mirnas as follows : ( i ) upregulated mirnas in both u937/nef - eyfp cells and exosomes ; ( ii ) downregulated mirnas in u937/nef - eyfp cells and upregulated mirnas in u937/nef - eyfp exosomes ; ( iii ) upregulated mirnas in u937/nef - eyfp cells and downregulated mirnas in u937/nef - eyfp exosomes . we then examined mirnas upregulated in both cellular and exosomal pools of u937/nef - eyfp compared to u937/eyfp cells ( mincupmineup ) . there were 87 and 311 in nef - expressing cells and exosomes , respectively , of which 77 mirnas were common to these groups ( fig . these 77 mirnas are likely to be packaged more in nef exosomes because their levels are also higher in nef - expressing cells . to identify mirnas selectively secreted in nef exosomes ( minesel ) we compared the mirnas down regulated in nef - expressing cells with those that were upregulated in nef exosomes ( mincdownnmineup ) and found 47 mirnas in this category ( fig . 2cii , supplementary file ) . finally , we analysed mirnas that are selectively retained in nef - expressing cells ( mincsel ) and are not exported out through exosomes . for this , we compared the group of mirnas upregulated in nef - expressing cells with those that were downregulated in nef exosomes ( mincupminedown ) , and found only 2 such mirnas ( fig . 2ciii , supplementary file ) . thus , nef expression leads to the selective secretion of many more mirnas in exosomes compared to those that are selectively retained in nef - expressing cells . to confirm the profiling data , we selected 13 mirnas for validation by stem - loop quantitative rt - pcr in multiple replicates of cells and exosomes . these include mir-573 and mir-638 ( upregulated in nef cells ) , mir-548d-3p and 564 ( same levels in nef and control cells ) , and mir-16 - 1 , mir-146a , mir-125b , mir-146b-3p , mir-181a , mir-27a , mir-570 , mir-610 and mir-624 * ( down regulated in nef cells ) . all these mirnas , except mir-146a , were upregulated in exosomes in the profiling array data . as there is no well documented control small rna for exosomes , we quantified rnu6 levels in both nef and control exosomes , and found these to be almost similar in multiple replicates of the 2 populations . the average ct values obtained were 23.3 in control exosomes and 23.7 in nef exosomes ( supplementary file ) . the results showed most of the mirna levels to correlate with the profiling data . in the cells we found 4 mirnas to show a trend opposite to the profiling data . these include mir-27a , which is upregulated , and mir-548d-3p , mir-573 and mir-638 , which are down regulated in nef - expressing cells based on qrt - pcr analysis ( fig . all the analysed mirnas showed higher levels in nef exosomes , which is consistent with the profiling data ( fig . total rna isolated from ( a ) cells and ( b ) exosomes was used to quantify expression levels of selected mirnas as described in methods . since each mirna can potentially target hundreds of transcripts , changes in their abundance can have significant effects on cellular pathways . the validated targets of mirnas that were selectively secreted from nef - expressing cells were extracted using the mirwalk software . a majority of these mirnas were found to target the expression of cytokines / chemokines ( e.g. interleukins ) , proteins involved in innate immunity ( e.g. interferon , nfkb , stats ) , tumour suppressors ( pten , rb1 ) , cell survival factors ( bcl2 , mcl1 ) and the hiv restriction factor apobec3 g . in monocytes , nef induces the synthesis of proinflammatory cytokines and chemokines . this effect is observed with nef expressed intracellularly as well as that added exogenously to monocytes ( 24 ) . il1 , il1 , il6 , tnf , mip1 , mip1 , which are induced in the presence of nef , and observed that many of the mirnas that target these cytokines , such as mir-146a , mir-146b-3p , mir-125b and mir-181a , are downregulated in nef - expressing cells , but are selectively secreted in exosomes from these cells . these 4 mirnas regulate proinflammatory cytokine levels , antigen processing and presentation pathways ( 25 ) . we quantified the expression levels of il1 , il1 , il6 , tnf , mip1 , mip1 , and il10 mrnas in u937/nef - eyfp and u937/eyfp cells . except for tnf , which did not show any significant change , all other mrnas were present at much higher levels in nef - expressing cells ( fig . the results showed > 10-fold increase for il1 , il6 and mip1 mrnas , and about 5-fold increase for il10 and mip1 mrnas . for 2 of these , il1 and il6 , the protein expression levels were also estimated by western blotting ; these were also higher in nef - expressing cells ( fig . ( a ) rna isolated from u937/nef - eyfp and u937/eyfp cells were tested for the expression of selected cytokine genes by qrt - pcr ; relative fold - changes were calculated from raw ct values using the data assist software ( abi ) . error bars represent standard errors from 3 experiments , each with triplicate measurements ; * p<0.05 . ( b ) western blots of lysates from u937/nef - eyfp and u937/eyfp cells for il1 and il6 levels . host mirnas can also target hiv transcripts leading to a reduced level of viral replication . we then analysed our profiling data to identify the status of mirnas that can potentially target the hiv genome . in silico analysis was performed with the 451 detected mirnas that were matched against 2 hiv-1 genomes  nl4 - 3 ( clade b ) and indiec1 ( clade c ) , using two separate software tools  microinspector and rnahybrid . of the 262 potential hiv-1 targeting mirnas identified through this process , 189 were also present in exosomes from the 2 cell lines . interestingly , 170 of these mirnas were upregulated and only 19 were downregulated in exosomes from u937/nef - eyfp compared to u937/eyfp cells ( fig . this overwhelming preference for the secretion of potentially hiv - targeting mirnas suggests that nef - induced vesicle secretion might be a unique viral strategy to selectively export virus targeting host mirnas . ( a ) diagram showing the correlation between hiv targeting mirnas and nef exosomal mirnas . all the detected mirnas were screened for potential binding sites on 2 different hiv-1 genomes ( nl4 - 3 and indiec1 ) using the microinspector algorithm ( http://bioinfo.uni-plovdiv.bg/microinspector/ ) ; the identified mirnas were verified using the rnahybrid algorithm ( http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/ ) . ( b ) mirnas present in exosomes that can potentially target the hiv-1 genome are separated based on whether their levels are higher or lower in nef compared to control exosomes . using mirwalk , we obtained 1,565 validated target genes for the 47 mirnas that were selectively secreted in nef exosomes ( minesel ; supplementary file ) and 107 validated target genes of the 2 mirnas that were selectively retained in nef - expressing cells ( mincsel ; supplementary file ) . these were then subjected to pathway analysis using the david ( database for annotation , visualization and integrated discovery ) bioinformatics resources . we identified 63 kegg pathways ( p<0.05 ) corresponding to the 1,565 validated targets of the 47 mirnas in minesel . these include tgf - beta signalling , toll - like receptor signalling , apoptosis , cell cycle , t - cell receptor signalling , p53 signalling , mitogen activated protein kinase ( mapk ) signalling , cytokine  cytokine receptor interaction , janus kinase - signal transducer and activator of transcription ( jak - stat ) signalling , b - cell receptor signalling , mtor signalling and chemokine signalling ( supplementary file ) . we also identified 26 kegg pathways ( p<0.05 ) corresponding to the 107 validated targets of the mirnas in mincsel . some important pathways here include cell cycle , mapk signalling , p53 signalling , t - cell receptor signalling , toll - like receptor signalling , cytokine  cytokine receptor interaction and regulation of actin cytoskeleton ( supplementary file ) . we further compared the entries reported in the hiv , human protein interaction database to be upregulated and downregulated by nef with the mirna targets identified in this study . this identified 19 proteins ( genes ) reported to be upregulated by nef to also be present in our set of 1,565 upregulated genes ; similarly , 2 proteins ( genes ) were in common with our set of 107 downregulated genes . the nef protein of hiv is expressed early during viral infection and is associated with cellular membranes , including endosomal membranes ( 2 ) . in addition to perturbing intracellular membranes and signalling , nef is secreted in exosomes ( 4 ) . interestingly , nef increases the formation of mvbs ( 26 ) , which are intracellular sites for exosome biogenesis , the secretion of these vesicles ( 27 ) , and interacts with the aip1/alix protein that is involved in mvb biogenesis ( 28 ) . consequently , nef promotes its own export through exosomes from different cell types and can induce apoptosis in bystander cd4 + t cells ( 3 , 4 ) . exosomes contain different proteins , mrnas and mirnas depending on their cell of origin ( 9 ) . they are taken up by recipient cells , in which the exosomal mrna is translated and the mirna can post - transcriptionally regulate gene expression ( 10 ) , thus supporting the view that exosomes function as intercellular messengers . modulation of exosomal mirnas is reported in several cancers as well as viral infections ( 29 ) . we have carried out the first mirnome analysis of nef - expressing human monocytic cells and their exosomes . we show that nef exosomes are enriched in mirnas that can target proinflammatory cytokines and other genes involved in key pathways like jak - stat signalling , mapk signalling and apoptosis . further , an overwhelming percentage of mirnas that can potentially target hiv-1 are secreted out of nef - expressing cells into exosomes . as reported earlier for other cell types , u937 cells expressing nef also showed increased exosome secretion . there was differential expression of about 50% of detected mirnas under the influence of nef . it was shown recently that reducing gw182 , which is an important component of glycine - tryptophan ( gw ) bodies , also reduced mirna secretion through exosomes ( 30 ) . this agrees with our recent findings that nef - expressing u937 cells have higher levels of gw182 ( 17 ) and display increased secretion of exosomes and mirnas reported here . we observed significant changes in the levels of several mirnas that regulate innate immune responses , especially the proinflammatory cytokines . these include mir-16 , mir-125b , mir-146a , mir-146b-3p and mir-181a , which are reduced in nef - expressing u937 cells . of these , mir-16 is required for the degradation of transcripts that contain au - rich elements in their 3utr , which includes mrnas for most of the inflammatory mediators . it regulates il6 , tnf and il8 , and high levels of mir-16 restrict the production of inflammatory mediators under non - stimulated conditions ( 25 ) . interestingly , mir-16 levels were ~50% lower in the nef cellular pool , suggesting that nef might regulate proinflammatory cytokines through this pathway . our profiling data showed about 3-fold reduction in mir-125b levels in nef - expressing u937 cells and a corresponding increase in their exosomes , suggesting that nef might also render cells more permissive for viral replication through mir-125b modulation . the mir-146 family comprises 2 members  mir146a and mir146b , of which the former is also a well - known inhibitor of innate immune responses ( 32 ) . its levels increase in myeloid cells in response to tlr2 , tlr4 or tlr5 , and exposure to inflammatory cytokines like tnf or il1 ( 33 , 34 ) ; in alveolar epithelial cells it reduces il1 levels ( 33 , 34 ) . we found mir-146a levels to be about 5-fold lower in u937/nef - eyfp cells compared to u937/eyfp cells , and observed a corresponding increase in nef exosomes . in thp-1 monocytic cells , mir-146a regulates inflammatory cytokines / chemokines , including il1 , il6 , tnf , il8 , ip10 , and mcp-1 ( 34 ) . high levels of mir-146a correlated with increased numbers of gw bodies in thp-1 cells , suggesting that gw bodies are markers for mirna activity during innate immune signalling ( 34 ) . our data showing reduced levels of mir-146a in nef - expressing monocytes further , mir-181a levels are also ~50% lower in u937/nef - eyfp cells compared to u937/eyfp cells , and show a corresponding increase in nef exosomes . this mirna regulates t - cell signalling by down regulating multiple phosphatases , and is thought to act as a rheostat that regulates protein phosphorylation levels ( 25 ) . thus , several mirnas that affect inflammatory cytokines and innate immune responses are present at reduced levels in nef - expressing monocytes and show a corresponding increase in exosomes secreted from these cells . correspondingly , the mrnas and expression of these cytokines / chemokines are at higher levels in nef - expressing cells . naturally then , modulation of host mirnas is emerging as a viral counter - strategy for successful infection ( 13 , 35 ) . hiv-1 infection of jurkat cells downregulates the polycistronic mirna cluster mir-17/92 , which includes mir-17/-18/-20a , mir-19a/-19b and mir-92a ( 16 ) . the downregulation of mir-21 , mir-26a , mir-29a , mir-29b and mir-29c was also observed in hiv patients ( 31 ) . we found mir-18 , mir-19a , mir-20a , mir-21 and mir-29b to be downregulated in nef expressing monocytes . a comparison of mirna expression patterns in resting and activated cd4 + t cells and use of specific antagomirs concluded that mir-28 , mir-125b , mir-150 , mir-223 and mir-382 target the nef/3ltr region and contribute to hiv latency in resting cd4 + t cells ; similar data were also reported for monocytes and macrophages ( 14 , 16 ) . our profiling demonstrated reduced levels of mir-125b and mir-223 in nef - expressing monocytes , whereas mir-382 was not detected . based on in silico analyses , mir-29a , mir-29b , mir-149 , mir-324 - 5p and mir-378 were reported to target conserved regions of the hiv-1 genome , including the nef gene ( 16 ) . of these , we found mir-29b to be downregulated in nef - expressing cells . a majority of mirnas that inhibit hiv replication , including mir-17 , mir-19a , mir-19b , mir-20a , mir-26a , mir-28 , mir-29a , mir-29b , mir-29c , mir-92a , mir-125b , mir-149 , mir-150 , mir-223 , mir-324 - 5p , mir-378 and mir-382 were present at 1.5-folds or higher levels in nef exosomes . further , when correlated with our in silico analysis of mirna target sites in the hiv genome , an overwhelming majority of mirnas that can potentially target hiv-1 genomes were present at increased levels in exosomes secreted by nef - expressing cells . we also found 47 mirnas to be present at increased levels in nef exosomes despite being present at reduced levels in nef - expressing cells . thus , nef expression reduces the cellular levels of several host mirnas that target innate immune responses and viral transcripts by exosome - mediated export . the increased formation of mvb membranes ( 26 , 28 ) and enhanced vesicle secretion ( 4 , 27 ) in the presence of nef can favour virus egress and spread . however , recent reports also show mvbs to be sites for mirna assembly and activity ( 36 , 37 ) . thus , the host can limit hiv replication by targeting viral transcripts to membranes , which it evolved to exploit . we have recently shown that nef attenuates the activity of let-7a , which silences target genes primarily at mvbs ( 17 ) . this is attributed to the ability of nef to interact with ago2 through 2 highly conserved gw motifs ( 17 ) . we now show that many mirnas whose levels are reduced in nef - expressing cells accumulate in exosomes secreted from these cells . thus , besides employing endosomal membranes for ago2 interaction , hiv nef further exploits the exosomal pathway to alter the cellular and exosomal distribution of mirnas . based on these findings , we propose that nef has evolved a 2-pronged suppressor function to inhibit mirna - mediated viral silencing and to modulate exosome - mediated cell - to - cell communication . how nef influences the selective expression and export of mirnas , and how the exosomal cargo influences uninfected cells remain important issues to explore in future studies . this work was funded by grants from the department of biotechnology ( dbt ) , government of india and the indian council of medical research ( icmr ) . a senior research fellowship from the council for scientific and industrial research ( csir ) , india , supported ma .