>TMEM209|NM_032842|3'UTR
   1 CAAGTCATATATTTAATTCTGACATTTAGACTATTTCACTGAACCAGAAGTCGAAACTAAACATCTCTGAGCCACTGACT
  81 CTTCTGAAATAAAATACACATGGGTGTATGTTACAGACTCTTTAGATTTAACAGAAAATGTAGCTGTTATGAAATGTAAT
 161 TGTAAAAATATGTCCCGTATCTTCTATATCGAGACATTGCCTTTAATTTTATATCGCTTTTCAGAAATTTCAGTTGACTA
 241 CAAAACTGCAACCCTTCGGATTTTTATTGACTCAAAATAGTGCCATTCCCCTTAATGAAATAGATTTTGAGTCTTTTTTT
 321 CATTGTAACCCCCAAATGAGAATCATCTACCTGATTCTTGTACCAAAAAAAAATTTTTTTCAGTCTTTTTTTTTTTTAGA
 401 GAGGGTCTCTGTCAACGCAGACTGGAGTGCAGTGGCACGATCTTAGCTCACTACAACTTCTGCCTCCCAGGCTCAAGCAA
 481 TTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCATGCACCACCACGCCCAGCTAATTTTTGTATTTTTAGTAG
 561 AGACAGGGTTTCACCATGTTGGCCAGGCTGGTCCCGAACTCCTGACCTCAGGTGATCCACCCACCTCGGCCTCCCAAAGT
 641 GCTGGGATTATAGGTGCGAGCCACTGCGCCCAGCCTCAGTTATTTTATTTAACAGTGTAAGTACTTAGAAAGTAAGAAAA
 721 TTGCTGATTTAGTTTTTTTTTTTTTTTTTTTAATGTTTCCCCAGCATTCTGCAAGTTAGGCCAAAGCCCACATTCAAAGA
 801 AATGAGATTAACTCTTAAGTAATGTATTAAGTTTATGTATATTTTAAAATATTATTAAAGGAGGTTTGAAAGTATTGACA
 881 TTTAAAAAGTCAACACTTAGATTAAATTTAGCTGGTAGTTTTAATTTGGGTTTTAGTTAAGAGTGTGAGGACATCAGGAA
 961 AACTGTTTACTACTTTGGTTTTAGCAGCTCAGTTTTACTATTCCATAATGTGTTATTTTTAAAGTTCTCTTTTTAAGATC
1041 ACAGTGATATCCTATCTTCAAATTTTTTAAATATGTTCAAAATATCTGGAGGGTGAGAAGTTACCAAGTTTGTATGTTTT
1121 GTTTGACTCACCATCTTTATTTTCTGTATATGTAGTAGCTGGCAATTGCATATATTTTCTTGATTAACATATTAGAGACT
1201 GCTTCCATCATCTTATGTAAACCTGGAAACAGCTGAAACTAGTCTTTTCTGAAGAACCGTGTATCAGTGTTAGATGTGCA
1281 TCCCGTTTTGTCATTCCCTCAGACTTTGAATACAGTCATTACTCTCTGGAAGAGAAATGTAAGTATATTTTTTGTTATCT
1361 GCAGTATGGTTTAACATGTATTAATAATACACATATGCAGACTCACTAAAGTATCCCCAGTAATTAGTAAATTCCAAATT
1441 TAAGTCAGTACAGTTCTGTATTTGTAAATTCTGTCTTTCAAAAAGTTGTTCGTGTTTTTAAAAGTTGATGTCCAAAGGAA
1521 AGGAGCTTCTTTTAAGTACTTTTGGTGTATGTTGAATATTTATTTGCTATGATATTCTAGAAGAAAATATCAAACATGTT
1601 AAATATAAGAACATGTTAAAATTTAAATTTGCTATTTCAAAAGTTACTGAAGATGTCTTGGAATAACAATTGTCATGCTG
1681 TTAAACTAGTTGCATATACTTCATTAAAAGCACCATGGAGAAACATTTTAACAATGAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' aaGGUGACGAUGUUGGAGGGUCu 5' |:|| || | :|||||||| Target 5' caCTACAACTTCTGCCTCCCAGg 3'	449 - 471	165.00	-23.60
2	miRNA 3' aaGGUGACGAUGUUGGAGGGUcu 5' |||| || |::||||||| Target 5' acCCAC--CT-CGGCCTCCCAaa 3'	619 - 638	144.00	-21.10
3	miRNA 3' aaGGUGACGAUGUUGGAGGG-UCu 5' |: |||| ||:|||||: || Target 5' ttCTCCTGCCTCAGCCTCCTGAGt 3'	481 - 504	125.00	-22.70

Mutant ID	Mutant Position	Mutant Source
COSN14259578	53	COSMIC
COSN30127339	96	COSMIC
COSN5647799	295	COSMIC
COSN26881708	381	COSMIC
COSN17075741	417	COSMIC
COSN15720460	601	COSMIC
COSN2201334	1397	COSMIC
COSN32057796	1549	COSMIC
COSN21393209	1583	COSMIC
COSN8352725	1670	COSMIC
COSN20091694	1686	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs372029714	6	dbSNP
rs759910565	9	dbSNP
rs1368670124	19	dbSNP
rs1459268062	20	dbSNP
rs576315280	24	dbSNP
rs192388531	27	dbSNP
rs1343046541	30	dbSNP
rs1157566420	33	dbSNP
rs1039004601	35	dbSNP
rs1382122510	37	dbSNP
rs536176063	44	dbSNP
rs1453578501	46	dbSNP
rs1369848334	50	dbSNP
rs941042258	52	dbSNP
rs781114246	53	dbSNP
rs1049537527	63	dbSNP
rs1405360727	70	dbSNP
rs1487961634	91	dbSNP
rs1283784446	96	dbSNP
rs1242159774	97	dbSNP
rs1412818233	104	dbSNP
rs565992702	110	dbSNP
rs1355003314	112	dbSNP
rs955163031	120	dbSNP
rs547359585	127	dbSNP
rs922451361	140	dbSNP
rs1227982757	149	dbSNP
rs577354076	150	dbSNP
rs1282105104	155	dbSNP
rs1305000563	164	dbSNP
rs187699708	174	dbSNP
rs956037904	176	dbSNP
rs770766983	177	dbSNP
rs1204299234	178	dbSNP
rs1250018147	179	dbSNP
rs925910316	190	dbSNP
rs1200486901	197	dbSNP
rs1012050035	200	dbSNP
rs1477102104	206	dbSNP
rs1169697547	215	dbSNP
rs184472546	216	dbSNP
rs1421983528	220	dbSNP
rs1360637126	257	dbSNP
rs193144576	258	dbSNP
rs1367142568	265	dbSNP
rs866295673	268	dbSNP
rs1421732717	273	dbSNP
rs1415505905	279	dbSNP
rs1302222063	280	dbSNP
rs1182294636	288	dbSNP
rs1477381445	301	dbSNP
rs1260686278	302	dbSNP
rs1340727254	322	dbSNP
rs1012309998	323	dbSNP
rs1272954829	325	dbSNP
rs1056351473	330	dbSNP
rs1488547973	334	dbSNP
rs1261436839	345	dbSNP
rs1268506092	351	dbSNP
rs960767061	357	dbSNP
rs1220887490	362	dbSNP
rs1035487544	368	dbSNP
rs1450032147	371	dbSNP
rs994367065	373	dbSNP
rs1047731485	374	dbSNP
rs907988000	374	dbSNP
rs930656619	385	dbSNP
rs897278313	387	dbSNP
rs1387010808	390	dbSNP
rs1425111120	397	dbSNP
rs1162674736	398	dbSNP
rs1236652536	398	dbSNP
rs1318414763	398	dbSNP
rs766149383	398	dbSNP
rs1325297638	400	dbSNP
rs532380215	404	dbSNP
rs965022365	404	dbSNP
rs912056573	405	dbSNP
rs1265185349	411	dbSNP
rs138641681	416	dbSNP
rs561169987	417	dbSNP
rs1285201111	420	dbSNP
rs955111054	431	dbSNP
rs1030728225	432	dbSNP
rs1402455280	438	dbSNP
rs1445118230	439	dbSNP
rs999162602	451	dbSNP
rs1366434260	456	dbSNP
rs889492590	460	dbSNP
rs1292506869	467	dbSNP
rs968129033	469	dbSNP
rs1351059051	479	dbSNP
rs549195812	480	dbSNP
rs1455228817	490	dbSNP
rs1049404301	492	dbSNP
rs1385657269	493	dbSNP
rs1453925837	496	dbSNP
rs1021997443	511	dbSNP
rs933756028	520	dbSNP
rs900934755	524	dbSNP
rs1339137543	526	dbSNP
rs1012574897	527	dbSNP
rs527868930	529	dbSNP
rs1328726921	532	dbSNP
rs1206930402	537	dbSNP
rs1265041872	538	dbSNP
rs1464743185	541	dbSNP
rs1199751889	546	dbSNP
rs560881695	548	dbSNP
rs1420692251	554	dbSNP
rs934514613	565	dbSNP
rs925771131	591	dbSNP
rs978740082	594	dbSNP
rs1195545272	595	dbSNP
rs948620947	596	dbSNP
rs916608931	611	dbSNP
rs1177972180	612	dbSNP
rs1406101478	624	dbSNP
rs1468821175	625	dbSNP
rs1478129134	627	dbSNP
rs545375346	628	dbSNP
rs1393549973	631	dbSNP
rs1190740041	637	dbSNP
rs960669722	641	dbSNP
rs1350136181	643	dbSNP
rs1267225292	650	dbSNP
rs1036563842	657	dbSNP
rs187989495	658	dbSNP
rs1317501836	660	dbSNP
rs1481544840	666	dbSNP
rs577973608	667	dbSNP
rs886498690	668	dbSNP
rs1048279655	669	dbSNP
rs1201205751	675	dbSNP
rs554196023	678	dbSNP
rs930627512	679	dbSNP
rs1173316345	691	dbSNP
rs920581472	692	dbSNP
rs1463945988	713	dbSNP
rs1299106362	717	dbSNP
rs1370279308	721	dbSNP
rs1039071462	726	dbSNP
rs534505267	729	dbSNP
rs1385531024	731	dbSNP
rs1397307217	732	dbSNP
rs1369144608	733	dbSNP
rs562831752	734	dbSNP
rs961602905	735	dbSNP
rs1309187988	736	dbSNP
rs1017518959	737	dbSNP
rs1273808469	738	dbSNP
rs1339884064	738	dbSNP
rs544732536	739	dbSNP
rs1397634424	743	dbSNP
rs1172638326	751	dbSNP
rs11435679	752	dbSNP
rs11459713	752	dbSNP
rs1188984092	752	dbSNP
rs1263142053	752	dbSNP
rs1430826573	752	dbSNP
rs1487927027	752	dbSNP
rs200514294	752	dbSNP
rs987578315	752	dbSNP
rs1291870659	753	dbSNP
rs1364242184	754	dbSNP
rs1462290137	754	dbSNP
rs940754590	755	dbSNP
rs1173868118	759	dbSNP
rs1362916806	760	dbSNP
rs1247927664	764	dbSNP
rs1434794029	765	dbSNP
rs1292181064	766	dbSNP
rs890029620	790	dbSNP
rs1269626919	795	dbSNP
rs1194309673	797	dbSNP
rs1447704741	797	dbSNP
rs1218853762	814	dbSNP
rs1262111832	820	dbSNP
rs1485949290	824	dbSNP
rs1187358004	837	dbSNP
rs1248007405	839	dbSNP
rs934835220	845	dbSNP
rs746583768	849	dbSNP
rs923660434	858	dbSNP
rs1028552001	867	dbSNP
rs978240740	869	dbSNP
rs1236570505	871	dbSNP
rs997870100	874	dbSNP
rs1163028213	875	dbSNP
rs1347151532	877	dbSNP
rs1457881654	912	dbSNP
rs1200697429	939	dbSNP
rs1340508140	943	dbSNP
rs1386399477	957	dbSNP
rs1454446212	964	dbSNP
rs1315488409	968	dbSNP
rs759394860	972	dbSNP
rs1378248682	973	dbSNP
rs1255730464	977	dbSNP
rs776637805	987	dbSNP
rs1052907844	988	dbSNP
rs1308752005	990	dbSNP
rs1317121272	996	dbSNP
rs1207586000	1000	dbSNP
rs1314435044	1001	dbSNP
rs934403697	1003	dbSNP
rs1463943259	1004	dbSNP
rs370252026	1007	dbSNP
rs1238551631	1008	dbSNP
rs1472697645	1012	dbSNP
rs1303557211	1027	dbSNP
rs544779461	1044	dbSNP
rs904378572	1047	dbSNP
rs1043267537	1050	dbSNP
rs183088489	1056	dbSNP
rs555875443	1061	dbSNP
rs915845555	1068	dbSNP
rs1470990908	1080	dbSNP
rs1334832112	1081	dbSNP
rs992663065	1083	dbSNP
rs1358475452	1086	dbSNP
rs1391539256	1088	dbSNP
rs1401266543	1106	dbSNP
rs1299625890	1116	dbSNP
rs207468606	1117	dbSNP
rs1373607637	1128	dbSNP
rs959249069	1131	dbSNP
rs1034952068	1135	dbSNP
rs145065892	1148	dbSNP
rs1395612705	1151	dbSNP
rs1325146490	1152	dbSNP
rs1174406599	1154	dbSNP
rs1439182595	1159	dbSNP
rs1223557723	1162	dbSNP
rs1280011594	1164	dbSNP
rs927949165	1172	dbSNP
rs1296758959	1179	dbSNP
rs554955151	1184	dbSNP
rs1234892506	1189	dbSNP
rs1483513113	1195	dbSNP
rs1181806583	1197	dbSNP
rs972883549	1200	dbSNP
rs1481484690	1208	dbSNP
rs961743158	1209	dbSNP
rs1378486627	1213	dbSNP
rs1017132460	1215	dbSNP
rs1026180362	1216	dbSNP
rs1412615070	1222	dbSNP
rs1172284105	1223	dbSNP
rs984302801	1226	dbSNP
rs954171543	1235	dbSNP
rs572135769	1244	dbSNP
rs998404317	1246	dbSNP
rs900875989	1258	dbSNP
rs1199062195	1259	dbSNP
rs1273689960	1261	dbSNP
rs1484564450	1267	dbSNP
rs1281648487	1280	dbSNP
rs376254994	1284	dbSNP
rs746588903	1285	dbSNP
rs1343403708	1291	dbSNP
rs370926197	1294	dbSNP
rs1350864479	1315	dbSNP
rs998642822	1317	dbSNP
rs1249955858	1318	dbSNP
rs1490889080	1323	dbSNP
rs1039431363	1331	dbSNP
rs1310754998	1340	dbSNP
rs1239340653	1348	dbSNP
rs1477070936	1354	dbSNP
rs1188254321	1357	dbSNP
rs904283732	1358	dbSNP
rs1310401878	1383	dbSNP
rs772940496	1387	dbSNP
rs1375776919	1395	dbSNP
rs1166421005	1396	dbSNP
rs1297513347	1402	dbSNP
rs1042762027	1406	dbSNP
rs1417746257	1411	dbSNP
rs1322330593	1416	dbSNP
rs1328898226	1422	dbSNP
rs1432248723	1427	dbSNP
rs890669689	1428	dbSNP
rs1012704448	1431	dbSNP
rs1228138586	1435	dbSNP
rs894277555	1439	dbSNP
rs1272844559	1444	dbSNP
rs1358771606	1451	dbSNP
rs1057008606	1460	dbSNP
rs934950031	1464	dbSNP
rs756282632	1477	dbSNP
rs1195401392	1481	dbSNP
rs771443559	1486	dbSNP
rs924782907	1492	dbSNP
rs554080182	1493	dbSNP
rs1186478911	1509	dbSNP
rs938600995	1518	dbSNP
rs946446767	1526	dbSNP
rs1472990583	1535	dbSNP
rs928013174	1538	dbSNP
rs915010953	1542	dbSNP
rs1388125110	1544	dbSNP
rs538507729	1545	dbSNP
rs1401995129	1547	dbSNP
rs570918760	1549	dbSNP
rs1185939358	1551	dbSNP
rs909976064	1559	dbSNP
rs1476398151	1566	dbSNP
rs984166498	1569	dbSNP
rs954202641	1570	dbSNP
rs1283886191	1605	dbSNP
rs555713559	1606	dbSNP
rs1461946842	1620	dbSNP
rs982017638	1633	dbSNP
rs921428982	1634	dbSNP
rs977025269	1640	dbSNP
rs1378770505	1655	dbSNP
rs965999630	1656	dbSNP
rs537428485	1660	dbSNP
rs1031892471	1669	dbSNP
rs747697831	1670	dbSNP
rs998944375	1672	dbSNP
rs968372153	1675	dbSNP
rs1244859395	1677	dbSNP
rs1442854014	1680	dbSNP
rs779372589	1686	dbSNP
rs1287597882	1694	dbSNP
rs1205205061	1696	dbSNP
rs994706951	1712	dbSNP
rs1410654218	1717	dbSNP
rs899214494	1718	dbSNP
rs1319217956	1725	dbSNP
rs144819368	1730	dbSNP
rs543924099	1730	dbSNP
rs1396988629	1737	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.