>STOML3|NM_001144033|3'UTR
   1 GGTCCTCTTGCGGTAGTCAGCTATTGCATAGAGAGTGGTGTCTATTCCTATGGAGAAGCCAGTAGTTAGAAACGGTGAGA
  81 ATTCCAACACTGTTCATGTAACAGTAACTCCATAGATGGAAGAGCATTGGCAGGAAAAAGCTCTAGAGGCTATACAGAGA
 161 AGAAAAAAAGAGCAAAAATGAGACCTGTATATTACTTTTATTTTTAGGACCATAATCAACTTTGTAATATTCTTATATAA
 241 TTAATTAGTGGTGGTCTCCGACTTTATAGCAGGAACTTTCTGGTAGTATGAGAAGAAATCATTGGGTGACTATGACTAAT
 321 GTTTCAGAATTACTTTTTAATTGTTTTAAAAAGCACTCCAGATAATTTATAGAGTAACATAGGCCTCATATTTACACTAT
 401 ATGTTCGCTTTATAATTCCGAAGTTGTTATTTGCTAAATCTCCAATGGTTGCAATCAAACCCCACAATGCTGAAAACAGA
 481 GATTGATTTTGATGTTAAGAACAAAAGAAGTATGGAAACTTCCAGGCTCAAACATGTCCCCCGTGCCACAAGGCAAACTG
 561 CGATGAGCTCACAGTCCAGGTCTCTTGCTGTGTGTCCTGCAGGGAGTGGCCAACTCTCCACCTTCCAGCACCATTATCAG
 641 AGGGTGGGAAAAGGCACATGAAAGTGGCCAGCTCCTGTCAATTTATTTGCTTACAAGGGAGAATGCTTGGAAAGGTTGCA
 721 TTGGTTGAAACCACTGAGGAGCATCACCCTGGGGATTTCATGTTTTCACTCTGTTCTTAGTCCCAGGAGCCACAGGTAAT
 801 CGAGAATGTTTGTGAACTCTATCCACATCCCCTCCCCCACCTGCCAAATAACCTGTTCAAAACAAAGAAGGCATAGAATA
 881 TAAATGAATTTCAAAAGTTAAAAAAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' aaACCAGUGUGGUCAGGUGUAa 5' || || |:| ||||||| Target 5' ttTGTGAACTCTA-TCCACATc 3'	809 - 829	151.00	-12.00
2	miRNA 3' aaACCAGUGUGGUCAGGUGUAa 5' ||| || ||: ||||| | Target 5' agTGGCCA-ACT-CTCCACCTt 3'	605 - 624	122.00	-9.42
3	miRNA 3' aaACCAGU--GUGGUC----AGGUGUaa 5' || ||| :| ||| ||||:| Target 5' acTGTTCATGTAACAGTAACTCCATAga 3'	89 - 116	120.00	-9.60

Mutant ID	Mutant Position	Mutant Source
COSN30714290	11	COSMIC
COSN20649797	25	COSMIC
COSN28680779	31	COSMIC
COSN30190547	31	COSMIC
COSN26997298	42	COSMIC
COSN30180592	48	COSMIC
COSN30111884	79	COSMIC
COSN30516043	79	COSMIC
COSN31604139	104	COSMIC
COSN30179187	131	COSMIC
COSN8587557	147	COSMIC
COSN24454871	155	COSMIC
COSN28878268	163	COSMIC
COSN20110620	372	COSMIC
COSN28197987	645	COSMIC
COSN29059373	718	COSMIC
COSN23987259	838	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1408248192	5	dbSNP
rs369471573	11	dbSNP
rs563608880	12	dbSNP
rs566858720	16	dbSNP
rs1411685840	21	dbSNP
rs375113273	23	dbSNP
rs1423129751	29	dbSNP
rs770745927	33	dbSNP
rs1480718494	34	dbSNP
rs1431231614	36	dbSNP
rs746600527	38	dbSNP
rs772932289	39	dbSNP
rs770376465	42	dbSNP
rs771470258	44	dbSNP
rs747764737	45	dbSNP
rs755594311	47	dbSNP
rs570615584	50	dbSNP
rs1324825650	51	dbSNP
rs903801239	52	dbSNP
rs1042310651	58	dbSNP
rs114077904	59	dbSNP
rs1328311405	64	dbSNP
rs111993514	73	dbSNP
rs1302970619	74	dbSNP
rs1348862835	81	dbSNP
rs1057432650	84	dbSNP
rs1259308287	88	dbSNP
rs866857341	92	dbSNP
rs938820435	93	dbSNP
rs908281619	95	dbSNP
rs927363799	97	dbSNP
rs1452043707	105	dbSNP
rs1192454685	108	dbSNP
rs1238060453	117	dbSNP
rs1455284923	125	dbSNP
rs1212356466	134	dbSNP
rs1156732418	139	dbSNP
rs1344412536	146	dbSNP
rs559513036	148	dbSNP
rs1290568420	150	dbSNP
rs1440830408	155	dbSNP
rs1438884348	156	dbSNP
rs1279878332	157	dbSNP
rs1229466552	159	dbSNP
rs1325264306	161	dbSNP
rs1344728805	162	dbSNP
rs1372062921	165	dbSNP
rs866308659	166	dbSNP
rs1236798332	168	dbSNP
rs1305080494	169	dbSNP
rs1316697053	170	dbSNP
rs1240132777	172	dbSNP
rs1263074100	179	dbSNP
rs7331403	184	dbSNP
rs952478761	186	dbSNP
rs920905793	189	dbSNP
rs1469383572	199	dbSNP
rs1175151498	201	dbSNP
rs1406705502	212	dbSNP
rs931465355	225	dbSNP
rs975111078	226	dbSNP
rs965983073	241	dbSNP
rs920071531	243	dbSNP
rs972972630	248	dbSNP
rs1019901620	250	dbSNP
rs751987434	253	dbSNP
rs1326441313	254	dbSNP
rs529259155	259	dbSNP
rs1021703074	260	dbSNP
rs1296677210	267	dbSNP
rs1011637150	268	dbSNP
rs1373134028	272	dbSNP
rs986869041	284	dbSNP
rs1333404834	285	dbSNP
rs894577453	291	dbSNP
rs1056160396	308	dbSNP
rs562093997	313	dbSNP
rs1336312640	318	dbSNP
rs1002884275	320	dbSNP
rs1232011729	324	dbSNP
rs1372131210	348	dbSNP
rs146739669	349	dbSNP
rs1178777888	358	dbSNP
rs1046322926	362	dbSNP
rs200079088	372	dbSNP
rs66601384	373	dbSNP
rs746531526	373	dbSNP
rs200622032	375	dbSNP
rs1474734654	377	dbSNP
rs543660306	380	dbSNP
rs967889284	381	dbSNP
rs1419863530	383	dbSNP
rs1020769423	387	dbSNP
rs939843794	388	dbSNP
rs1168412857	390	dbSNP
rs908355575	399	dbSNP
rs1048235101	400	dbSNP
rs1035730395	406	dbSNP
rs931026852	407	dbSNP
rs577823866	419	dbSNP
rs1414477610	425	dbSNP
rs1003273941	430	dbSNP
rs975479328	440	dbSNP
rs1044480209	444	dbSNP
rs1474251885	444	dbSNP
rs557894604	445	dbSNP
rs898675858	446	dbSNP
rs1206078275	447	dbSNP
rs912785563	452	dbSNP
rs1272851168	461	dbSNP
rs748150667	464	dbSNP
rs1037123097	486	dbSNP
rs879705342	491	dbSNP
rs879264292	492	dbSNP
rs1021776551	497	dbSNP
rs990318659	501	dbSNP
rs185041055	503	dbSNP
rs986510537	526	dbSNP
rs9532314	529	dbSNP
rs555153932	530	dbSNP
rs535474270	531	dbSNP
rs180695238	538	dbSNP
rs1313867424	539	dbSNP
rs1296154964	542	dbSNP
rs907230110	543	dbSNP
rs1270086263	545	dbSNP
rs1014891488	547	dbSNP
rs1229892184	553	dbSNP
rs960687348	561	dbSNP
rs1035332493	562	dbSNP
rs1004063205	564	dbSNP
rs1282173709	565	dbSNP
rs1350298304	567	dbSNP
rs887029499	568	dbSNP
rs1242568241	570	dbSNP
rs1475790871	572	dbSNP
rs544027419	598	dbSNP
rs1459832301	603	dbSNP
rs7990956	609	dbSNP
rs745812228	612	dbSNP
rs578103571	637	dbSNP
rs7989622	638	dbSNP
rs1176878694	640	dbSNP
rs533717167	647	dbSNP
rs1318262290	652	dbSNP
rs1488998872	654	dbSNP
rs1436724838	656	dbSNP
rs530573803	658	dbSNP
rs764912135	670	dbSNP
rs1369848901	677	dbSNP
rs1234601206	698	dbSNP
rs566176929	704	dbSNP
rs946240725	707	dbSNP
rs914833925	713	dbSNP
rs1334769220	716	dbSNP
rs940169610	719	dbSNP
rs1466197330	720	dbSNP
rs990308870	724	dbSNP
rs1210717457	733	dbSNP
rs41286137	734	dbSNP
rs1034835313	736	dbSNP
rs529337831	741	dbSNP
rs1228460169	743	dbSNP
rs41286135	748	dbSNP
rs1194508302	750	dbSNP
rs971907935	769	dbSNP
rs1379530505	776	dbSNP
rs528102209	783	dbSNP
rs1158344904	786	dbSNP
rs149173223	787	dbSNP
rs558474528	793	dbSNP
rs887019690	796	dbSNP
rs777260620	798	dbSNP
rs564412643	801	dbSNP
rs145177657	802	dbSNP
rs1333607122	804	dbSNP
rs1441298592	817	dbSNP
rs1438541694	818	dbSNP
rs1331203807	820	dbSNP
rs1393663237	834	dbSNP
rs1272398110	835	dbSNP
rs6563658	838	dbSNP
rs1458858549	852	dbSNP
rs988016888	867	dbSNP
rs370083275	874	dbSNP
rs1473755417	875	dbSNP
rs960844038	876	dbSNP
rs189323858	878	dbSNP
rs927792807	886	dbSNP
rs752213138	894	dbSNP

- mBio, 2013

UNLABELLED: The question of how HIV-1 interfaces with cellular microRNA (miRNA) biogenesis and effector mechanisms has been highly controversial. Here, we first used deep sequencing of small RNAs present in two different infected cell lines (TZM-bl and C8166) and two types of primary human cells (CD4(+) peripheral blood mononuclear cells [PBMCs] and macrophages) to unequivocally demonstrate that HIV-1 does not encode any viral miRNAs. Perhaps surprisingly, we also observed that infection of T cells by HIV-1 has only a modest effect on the expression of cellular miRNAs at early times after infection. Comprehensive analysis of miRNA binding to the HIV-1 genome using the photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) technique revealed several binding sites for cellular miRNAs, a subset of which were shown to be capable of mediating miRNA-mediated repression of gene expression. However, the main finding from this analysis is that HIV-1 transcripts are largely refractory to miRNA binding, most probably due to extensive viral RNA secondary structure. Together, these data demonstrate that HIV-1 neither encodes viral miRNAs nor strongly influences cellular miRNA expression, at least early after infection, and imply that HIV-1 transcripts have evolved to avoid inhibition by preexisting cellular miRNAs by adopting extensive RNA secondary structures that occlude most potential miRNA binding sites. IMPORTANCE: MicroRNAs (miRNAs) are a ubiquitous class of small regulatory RNAs that serve as posttranscriptional regulators of gene expression. Previous work has suggested that HIV-1 might subvert the function of the cellular miRNA machinery by expressing viral miRNAs or by dramatically altering the level of cellular miRNA expression. Using very sensitive approaches, we now demonstrate that neither of these ideas is in fact correct. Moreover, HIV-1 transcripts appear to largely avoid regulation by cellular miRNAs by adopting an extensive RNA secondary structure that occludes the ability of cellular miRNAs to interact with viral mRNAs. Together, these data suggest that HIV-1, rather than seeking to control miRNA function in infected cells, has instead evolved a mechanism to become largely invisible to cellular miRNA effector mechanisms.