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 .
 introductionthe hiv nef protein is a multifunctional virulence factor that perturbs intracellular membranes and signalling and is secreted into exosomes . while nef - containing exosomes have a distinct proteomic profile , no comprehensive analysis of their mirna cargo has been carried out . 
 since nef functions as a viral suppressor of rna interference and disturbs the distribution of rna - induced silencing complex proteins between cells and exosomes , we hypothesized that it might also affect the export of mirnas into exosomes.methodexosomes were purified from human monocytic u937 cells that stably expressed hiv-1 nef . 
 the rna from cells and exosomes was profiled for 667 mirnas using a taqman low density array . selected mirnas and their mrna targets 
 were validated by quantitative rt - pcr . 
 bioinformatics analyses were used to identify targets and predict pathways.resultsnef expression affected a significant fraction of mirnas in u937 cells . 
 our analysis showed 47 mirnas to be selectively secreted into nef exosomes and 2 mirnas to be selectively retained in nef - expressing cells . 
 the exosomal mirnas were predicted to target several cellular genes in inflammatory cytokine and other pathways important for hiv pathogenesis , and an overwhelming majority had targets within the hiv genome.conclusionsthis is the first study to report mirnome analysis of hiv nef expressing monocytes and exosomes . 
 our results demonstrate that nef causes large - scale dysregulation of cellular mirnas , including their secretion through exosomes . 
 we suggest this to be a novel viral strategy to affect pathogenesis and to limit the effects of rna interference on viral replication and persistence . 
Materials and methods
Exosome isolation and characterization
Isolation of cellular and exosomal RNAs
Profiling of miRNAs in cells and exosomes
Quantification of miRNAs and mRNAs
Bioinformatic analysis
Western blotting
Results
Characterization of exosomes secreted from U937 human monocytic cells
Differential miRNA expression and secretion in exosomes
Validation of miRNAs in cells and exosomes
Nef exosomes are enriched in miRNAs that potentially target innate immune responses and 
the HIV-1 genome
Pathway analysis of selectively deregulated miRNAs in Nef expressing cells and exosomes
Discussion
Conflict of interest and funding