>BBC3|NM_001127241|3'UTR
   1 GTGCCTGCACCCGCCCGGTGGACGTCAGGGACTCGGGGGGCAGGCCCCTCCCACCTCCTGACACCCTGGCCAGCGCGGGG
  81 GACTTTCTCTGCACCATGTAGCATACTGGACTCCCAGCCCTGCCTGTCCCGGGGGCGGGCCGGGGCAGCCACTCCAGCCC
 161 CAGCCCAGCCTGGGGTGCACTGACGGAGATGCGGACTCCTGGGTCCCTGGCCAAGAAGCCAGGAGAGGGACGGCTGATGG
 241 ACTCAGCATCGGAAGGTGGCGGTGACCGAGGGGGTGGGGACTGAGCCGCCCGCCTCTGCCGCCCACCACCATCTCAGGAA
 321 AGGCTGTTGTGCTGGTGCCCGTTCCAGCTGCAGGGGTGACACTGGGGGGGGGGGGCTCTCCTCTCGGTGCTCCTTCACTC
 401 TGGGCCTGGCCTCAGGCCCCTGGTGCTTCCCCCCCTCCTCCTGGGAGGGGGCCCGTGAAGAGCAAATGAGCCAAACGTGA
 481 CCACTAGCCTCCTGGAGCCAGAGAGTGGGGCTCGTTTGCCGGTTGCTCCAGCCCGGCGCCCAGCCATCTTCCCTGAGCCA
 561 GCCGGCGGGTGGTGGGCATGCCTGCCTCACCTTCATCAGGGGGTGGCCAGGAGGGGCCCAGACTGTGAATCCTGTGCTCT
 641 GCCCGTGACCGCCCCCCGCCCCATCAATCCCATTGCATAGGTTTAGAGAGAGCACGTGTGACCACTGGCATTCATTTGGG
 721 GGGTGGGAGATTTTGGCTGAAGCCGCCCCAGCCTTAGTCCCCAGGGCCAAGCGCTGGGGGGAAGACGGGGAGTCAGGGAG
 801 GGGGGGAAATCTCGGAAGAGGGAGGAGTCTGGGAGTGGGGAGGGATGGCCCAGCCTGTAAGATACTGTATATGCGCTGCT
 881 GTAGATACCGGAATGAATTTTCTGTACATGTTTGGTTAATTTTTTTTGTACATGATTTTTGTATGTTTCCTTTTCAATAA
 961 AATCAGATTGGAACAGTGGAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ugaaaGGGAAGU----CUCGGACCGa 5' :|||||| |:||||||| Target 5' ggtgcTCCTTCACTCTGGGCCTGGCc 3'	386 - 411	163.00	-23.50
2	miRNA 3' ugaaaGGGAAGUCUCGGACCga 5' ||| | ||||||| Target 5' ccagcCCCAGCCCAGCCTGGgg 3'	154 - 175	133.00	-18.20
3	miRNA 3' ugaaaGGGAAGUCUCGGACCGa 5' |:| | | | |||||| Target 5' cccacCTCCTGACACCCTGGCc 3'	50 - 71	125.00	-14.40

Mutant ID	Mutant Position	Mutant Source
770466	23	ClinVar
COSM7795714	9	COSMIC
COSM6945186	12	COSMIC
COSM7730930	13	COSMIC
COSM8230179	13	COSMIC
COSM6384941	16	COSMIC
COSM3892954	34	COSMIC
COSM5833783	34	COSMIC
COSM8156305	38	COSMIC
COSM8080915	46	COSMIC
COSM9481181	46	COSMIC
COSM8081793	47	COSMIC
COSM8185539	50	COSMIC
COSM7633355	57	COSMIC
COSM7659014	70	COSMIC
COSM8761196	75	COSMIC
COSM5973040	76	COSMIC
COSM9332578	78	COSMIC
COSM9481015	79	COSMIC
COSM8948125	101	COSMIC
COSN29589854	130	COSMIC
COSN513272	367	COSMIC
rs45474992	495	GWAS

Mutant ID	Mutant Position	Mutant Source
rs1425337655	4	dbSNP
rs769349350	7	dbSNP
rs747649341	9	dbSNP
rs1034388452	10	dbSNP
rs1237610094	11	dbSNP
rs184609559	12	dbSNP
rs755414185	13	dbSNP
rs752056442	15	dbSNP
rs780312516	16	dbSNP
rs758721081	17	dbSNP
rs202031784	23	dbSNP
rs765552886	24	dbSNP
rs1446034314	25	dbSNP
rs762024851	28	dbSNP
rs1194670852	32	dbSNP
rs199804290	34	dbSNP
rs764225454	35	dbSNP
rs759426766	37	dbSNP
rs774280756	40	dbSNP
rs1039339737	41	dbSNP
rs762488821	41	dbSNP
rs1257897798	46	dbSNP
rs1247197811	47	dbSNP
rs1342443339	48	dbSNP
rs770648214	52	dbSNP
rs1390883246	54	dbSNP
rs1232734535	56	dbSNP
rs762793628	57	dbSNP
rs888334888	59	dbSNP
rs772912619	60	dbSNP
rs568884204	61	dbSNP
rs548944939	64	dbSNP
rs990369562	67	dbSNP
rs1446231932	69	dbSNP
rs747474100	74	dbSNP
rs373478289	75	dbSNP
rs750787778	76	dbSNP
rs779328577	77	dbSNP
rs754077264	78	dbSNP
rs1277941099	79	dbSNP
rs1489988172	81	dbSNP
rs1338960527	82	dbSNP
rs775156041	82	dbSNP
rs927372009	82	dbSNP
rs764089253	86	dbSNP
rs1223595373	92	dbSNP
rs760753461	94	dbSNP
rs1259185140	95	dbSNP
rs1238658794	96	dbSNP
rs1336404997	97	dbSNP
rs970498389	98	dbSNP
rs1198233958	99	dbSNP
rs1403215618	106	dbSNP
rs1344727886	113	dbSNP
rs1488404253	115	dbSNP
rs1303703612	117	dbSNP
rs751511571	121	dbSNP
rs766332694	122	dbSNP
rs1157599708	123	dbSNP
rs1454036492	124	dbSNP
rs1365203637	125	dbSNP
rs1260724465	126	dbSNP
rs1025258331	127	dbSNP
rs1020481307	130	dbSNP
rs762694025	131	dbSNP
rs560015110	133	dbSNP
rs1453659061	135	dbSNP
rs1012064918	136	dbSNP
rs1382301354	136	dbSNP
rs989046061	137	dbSNP
rs894959866	138	dbSNP
rs1204943029	139	dbSNP
rs546445366	140	dbSNP
rs776198420	141	dbSNP
rs1221702836	142	dbSNP
rs1026873446	143	dbSNP
rs768062826	145	dbSNP
rs1438573409	146	dbSNP
rs142745657	147	dbSNP
rs746526541	150	dbSNP
rs533069381	151	dbSNP
rs144766692	159	dbSNP
rs996759116	160	dbSNP
rs1310781593	165	dbSNP
rs1353844777	166	dbSNP
rs1477214746	167	dbSNP
rs899543888	172	dbSNP
rs1018460117	174	dbSNP
rs1210209800	175	dbSNP
rs897165992	184	dbSNP
rs544099328	185	dbSNP
rs1249014258	186	dbSNP
rs1047008730	191	dbSNP
rs1240062223	193	dbSNP
rs1200156790	196	dbSNP
rs1440426422	198	dbSNP
rs1158218885	199	dbSNP
rs1362279569	202	dbSNP
rs1459698181	205	dbSNP
rs1164183238	206	dbSNP
rs1488040997	207	dbSNP
rs1439301866	210	dbSNP
rs1333988423	211	dbSNP
rs1376930423	214	dbSNP
rs1261106208	225	dbSNP
rs944149403	229	dbSNP
rs1354689364	231	dbSNP
rs891192064	232	dbSNP
rs531013232	251	dbSNP
rs1216993683	252	dbSNP
rs932817108	259	dbSNP
rs937189175	260	dbSNP
rs914708259	261	dbSNP
rs1200407491	265	dbSNP
rs927370782	267	dbSNP
rs1342574944	268	dbSNP
rs919762398	269	dbSNP
rs1458219709	270	dbSNP
rs1038132968	275	dbSNP
rs1178099408	277	dbSNP
rs990400673	278	dbSNP
rs1188559372	287	dbSNP
rs942646701	288	dbSNP
rs923638539	291	dbSNP
rs369262792	292	dbSNP
rs977775769	300	dbSNP
rs1465613218	303	dbSNP
rs970909080	304	dbSNP
rs1380760227	308	dbSNP
rs1396209194	318	dbSNP
rs1353263997	328	dbSNP
rs1024746360	337	dbSNP
rs1220237404	340	dbSNP
rs1278682746	341	dbSNP
rs1317213914	343	dbSNP
rs1202416194	346	dbSNP
rs1345445954	350	dbSNP
rs1174883984	355	dbSNP
rs1413156331	357	dbSNP
rs1427690191	362	dbSNP
rs1201203355	363	dbSNP
rs200154173	363	dbSNP
rs1168990396	364	dbSNP
rs1311918089	364	dbSNP
rs868724189	364	dbSNP
rs1212698283	365	dbSNP
rs1466112707	365	dbSNP
rs947372607	366	dbSNP
rs959206890	366	dbSNP
rs1185226395	367	dbSNP
rs2862236	367	dbSNP
rs1026735812	368	dbSNP
rs1368879676	368	dbSNP
rs769329459	368	dbSNP
rs1018003240	369	dbSNP
rs542241792	369	dbSNP
rs1432933137	370	dbSNP
rs888102385	371	dbSNP
rs986714376	371	dbSNP
rs1158816124	372	dbSNP
rs764799400	372	dbSNP
rs1398659875	373	dbSNP
rs897064631	373	dbSNP
rs1323829305	374	dbSNP
rs993835802	374	dbSNP
rs1057423547	375	dbSNP
rs10647392	376	dbSNP
rs1271844359	377	dbSNP
rs1422023467	377	dbSNP
rs1195972043	378	dbSNP
rs1488629666	379	dbSNP
rs1231871339	380	dbSNP
rs1237161284	382	dbSNP
rs1208278806	384	dbSNP
rs1468255265	385	dbSNP
rs1440545860	386	dbSNP
rs1280493917	388	dbSNP
rs1414187389	389	dbSNP
rs780394656	394	dbSNP
rs1460740310	398	dbSNP
rs1162617671	416	dbSNP
rs573226697	418	dbSNP
rs1001363710	428	dbSNP
rs1008246613	430	dbSNP
rs905716495	433	dbSNP
rs1043143687	434	dbSNP
rs1291896562	435	dbSNP
rs1199866792	436	dbSNP
rs373224142	436	dbSNP
rs1271630369	437	dbSNP
rs553411928	438	dbSNP
rs1211724093	451	dbSNP
rs1049842632	453	dbSNP
rs1474713421	454	dbSNP
rs1185506996	455	dbSNP
rs996893380	455	dbSNP
rs546484688	465	dbSNP
rs1445335908	472	dbSNP
rs1360473408	477	dbSNP
rs1054622050	482	dbSNP
rs933343788	489	dbSNP
rs45474992	495	dbSNP
rs1336329703	497	dbSNP
rs1376289680	505	dbSNP
rs924873198	506	dbSNP
rs975422267	513	dbSNP
rs1046383875	514	dbSNP
rs964903341	516	dbSNP
rs909402999	519	dbSNP
rs1173419851	520	dbSNP
rs949094401	521	dbSNP
rs557507372	531	dbSNP
rs990534054	534	dbSNP
rs751452575	535	dbSNP
rs375874652	537	dbSNP
rs1192313243	538	dbSNP
rs919788508	540	dbSNP
rs1479007359	547	dbSNP
rs1395823108	548	dbSNP
rs1001898189	563	dbSNP
rs973858382	566	dbSNP
rs961478943	567	dbSNP
rs1473902340	569	dbSNP
rs923927959	570	dbSNP
rs905688506	572	dbSNP
rs1015523469	575	dbSNP
rs1011369209	588	dbSNP
rs1008525843	599	dbSNP
rs1204090909	599	dbSNP
rs955401265	601	dbSNP
rs1028738009	603	dbSNP
rs1346569012	607	dbSNP
rs1231568683	609	dbSNP
rs1287737845	610	dbSNP
rs1354071324	615	dbSNP
rs1204329507	617	dbSNP
rs1256121061	619	dbSNP
rs1259470948	620	dbSNP
rs1453232310	627	dbSNP
rs1200446352	644	dbSNP
rs116590103	645	dbSNP
rs188608771	650	dbSNP
rs60480476	651	dbSNP
rs1380927751	652	dbSNP
rs999045422	657	dbSNP
rs903323275	658	dbSNP
rs1446572918	659	dbSNP
rs923303898	660	dbSNP
rs1369540904	663	dbSNP
rs1438087175	665	dbSNP
rs1045989766	667	dbSNP
rs1335970203	672	dbSNP
rs950310606	680	dbSNP
rs1237942509	693	dbSNP
rs943581111	695	dbSNP
rs917631863	703	dbSNP
rs1398554322	713	dbSNP
rs909469164	716	dbSNP
rs555221336	718	dbSNP
rs759995630	719	dbSNP
rs984933007	720	dbSNP
rs1467134481	723	dbSNP
rs1334019431	724	dbSNP
rs1240155728	739	dbSNP
rs937785050	744	dbSNP
rs919695415	745	dbSNP
rs973763679	749	dbSNP
rs961241699	752	dbSNP
rs1162809818	764	dbSNP
rs1407109313	768	dbSNP
rs1386152256	772	dbSNP
rs1460391121	773	dbSNP
rs1332769246	774	dbSNP
rs908330809	776	dbSNP
rs986773208	777	dbSNP
rs1396644361	783	dbSNP
rs774716923	784	dbSNP
rs1438923027	786	dbSNP
rs955462011	787	dbSNP
rs1233669884	789	dbSNP
rs535181853	791	dbSNP
rs1236118144	794	dbSNP
rs1028358931	796	dbSNP
rs1253108816	800	dbSNP
rs996833314	801	dbSNP
rs1001216586	802	dbSNP
rs970342323	805	dbSNP
rs1021563341	807	dbSNP
rs1218768729	808	dbSNP
rs1011337938	810	dbSNP
rs957337371	811	dbSNP
rs1033110066	813	dbSNP
rs566092441	814	dbSNP
rs1353361111	817	dbSNP
rs546205340	818	dbSNP
rs1220334036	825	dbSNP
rs766774982	849	dbSNP
rs1308086506	851	dbSNP
rs1226785810	855	dbSNP
rs533030651	864	dbSNP
rs1249752952	873	dbSNP
rs763271309	874	dbSNP
rs1211915526	875	dbSNP
rs1257208003	881	dbSNP
rs1484908938	884	dbSNP
rs569336172	888	dbSNP
rs550735487	889	dbSNP
rs530663306	890	dbSNP
rs1428501503	898	dbSNP
rs561922996	904	dbSNP
rs1378896472	910	dbSNP
rs1434904263	915	dbSNP
rs1172647967	928	dbSNP
rs1373326706	928	dbSNP
rs997589715	941	dbSNP
rs1289302083	942	dbSNP
rs74710244	947	dbSNP
rs1380509035	957	dbSNP
rs1293484351	964	dbSNP
rs1039405493	965	dbSNP
rs1235239286	967	dbSNP
rs943400826	972	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.