>SESN2|NM_031459|3'UTR
   1 CTCCTGAGCAGGACCTGGGCCCGGTTCAGCTCCCCACAAGGACTTCTCTGTCTGGAGACAGCCCCAGACCCTTTTGTGTC
  81 CCATGCCCACCCTCCCCACGCTGCAGTGGGCTTGTGTGTGATGTGCAGTCCCGAAGCCACACCCTCCCTTTTCCTCACTG
 161 GAATGGACAGTTCATTGCACTGACTCTGGGATCTCAGCCCTGCTCCTGGGAGCTGGAAGAGCACTTGGAGATCCTAAGGG
 241 ACCACACCCTTCCTCCTTCCCCTGCCCACAGAGGCAGAGGGCACAGGAAAGAAGCCGGGCCAAGCTCGGAATTAATGTGC
 321 CACAAGTGTTGTGGCCTTCCTGAACTGGGAAGTCCCTGGCTGGCCCCCGGGGGAGAGGGGCAAATGCCTCCGGGACTGAC
 401 ACTCCAGGCAGCTTTGCCTTCTCTCCCCTGTCATTTCCAGATTTCATTACCTCCTACTTGCCATTCACCCATCAATGTGA
 481 AAGTCAGGGTCACAGCTGGTCTGTGTGTCCAGTTCCCTAAAAGCCTGTTCTGTTGGGCAGCCTGAGGCTGTTGCCCGAAT
 561 CCTAGTTCAGTTTTTTGACTTCCTTTGCCCTTTTTCCCTTTTCTCCATGCTTAATGGTGTGAGGCGTCAGGAGAGAGGCC
 641 AAGTACATAAAAAAAAAAAAAGCAGATTATCTCTAGAGAGTTTGAGCCTTTGCTGGTCACATTGCCTTCTGAAGAGGAGG
 721 GAGTATTAGATTATAAATCCTCTTTATTTTGGTCCTTTATGCTTGAGGTTCCAACCTGGAGCCACAGTGTGTGAGAGGAG
 801 GAGGAGAGGGAGAATTCTGTTCTCCCAGAGCTGCACCTGCCTCGCAGAGGCCAGCACCCCACTCTCCTGCCTCCAGTGGC
 881 CCTGCCGCAGATGTCTCCCAAAAAGTTGAGCCTTTCTAGATGGCTTAGGTGGCACCATGGCTCAGCAGGAGGGGCGGGAG
 961 GCACCAGGGTTCTTGTTTGGACCCTGCCCCTGGGCCATGGCCAGGTGACCATGGCTACATTGCCAAACCTCTGACTGCCA
1041 CAGCTGCAGACTGAGAGGGTGGGTCTGAGTCCCCACAATGTCTGAAGCTGCCCCTGGGATTCTCAGGCCAACCTGCCAAC
1121 AGCAAGCGGATTTTCTTGCAAGATCAGGGACCCCATTTCTGCAGCCAGTGTCTCCTGGGTGCCTTCTGAGGACTCCCACC
1201 CCCATCCCAGTATCTCATCTGTCCCCTCTCCTGGGGCTTAAGTGGGTTGCTTCCAGGCAGAAGCAGCCAAGGACCGATTC
1281 CAGGCACTTTCTGTAGCAAATGACTGTGAATTACGACTTCTCTTGCCCTTCTTCTAGCAGTCTGTGCCTCCTCTCTGACC
1361 AGTTTGGAGGGCACTGAAGAAAGGCAAGGGCCGTGCTGCTGCTGGGCGGGGCAGGAGAGGAGCCTGGCCAGTGTGCCACA
1441 TTAAATACCCGTGCAGGCGCGGAGAAGCAACCGGCACCCCCTTCCGGCCTGAAAGCCCTCCCTGCAAGAAGGTGTGCAGG
1521 AGAGAAGAGGCCCCGGCATGGGGATCTGGGTTCTAGAGGGCATGTGATGACTGTAAATGTTCACTGGGTGGGTAGGGAGT
1601 GGTATCCAGTGTTCAAGTGCAGAAATCTTTGGCTTTGCTACCAGTTCCATATGATGAGAAATAAACGTTCGCTGAGGTTT
1681 TGTTTCATAAAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cuUGUCUAUCAGAU-UUGUGACcc 5' |::|| |||: | :||||| Target 5' gaATGGACAGTTCATTGCACTGac 3'	161 - 184	125.00	-12.00
2	miRNA 3' cuuguCUAUCAGAUU---UGU-GACCc 5' || |||| :: ||| |||| Target 5' aatgtGAAAGTCAGGGTCACAGCTGGt 3'	474 - 500	122.00	-10.00
3	miRNA 3' cuugucuaucagauuuGUGACCc 5' |||||| Target 5' gatgactgtaaatgttCACTGGg 3'	1566 - 1588	120.00	-14.20

Mutant ID	Mutant Position	Mutant Source
COSN26636402	20	COSMIC
COSN30508915	24	COSMIC
COSN30485371	95	COSMIC
COSN30027538	131	COSMIC
COSN5740424	188	COSMIC
COSN24325465	571	COSMIC
COSN1102439	662	COSMIC
COSN20094622	662	COSMIC
COSN17181560	787	COSMIC
COSN29796172	1091	COSMIC
COSN28202125	1180	COSMIC
COSN27800418	1536	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs769395715	4	dbSNP
rs547652634	5	dbSNP
rs370925800	10	dbSNP
rs766661062	18	dbSNP
rs757483688	23	dbSNP
rs375396916	24	dbSNP
rs767831374	30	dbSNP
rs752886912	31	dbSNP
rs758274242	32	dbSNP
rs1179983876	35	dbSNP
rs1257212704	38	dbSNP
rs756219286	40	dbSNP
rs777781076	41	dbSNP
rs1187940130	43	dbSNP
rs1261574951	44	dbSNP
rs779852360	46	dbSNP
rs961534163	49	dbSNP
rs1168889678	51	dbSNP
rs992991731	53	dbSNP
rs917436163	57	dbSNP
rs949163716	60	dbSNP
rs980393843	62	dbSNP
rs1291423893	68	dbSNP
rs1288225559	72	dbSNP
rs926281645	77	dbSNP
rs1401645582	79	dbSNP
rs950275639	81	dbSNP
rs936351159	87	dbSNP
rs1344520352	90	dbSNP
rs140893776	92	dbSNP
rs1221299098	100	dbSNP
rs533051045	101	dbSNP
rs1248383828	103	dbSNP
rs1053668986	115	dbSNP
rs892382959	119	dbSNP
rs1466119284	126	dbSNP
rs1181555670	133	dbSNP
rs549585881	134	dbSNP
rs1264304457	141	dbSNP
rs1480268657	147	dbSNP
rs116173499	165	dbSNP
rs887362317	174	dbSNP
rs1427454250	180	dbSNP
rs945181719	182	dbSNP
rs1040938240	190	dbSNP
rs1383565290	196	dbSNP
rs1421413970	198	dbSNP
rs1298653347	212	dbSNP
rs190182009	226	dbSNP
rs1402812583	232	dbSNP
rs1294929594	244	dbSNP
rs1367817449	257	dbSNP
rs1163844029	260	dbSNP
rs901239945	262	dbSNP
rs1038462090	272	dbSNP
rs899562098	290	dbSNP
rs1242940450	296	dbSNP
rs1399455413	297	dbSNP
rs996962059	298	dbSNP
rs17163103	304	dbSNP
rs1029488419	308	dbSNP
rs1488919449	321	dbSNP
rs1176733916	325	dbSNP
rs1236429357	346	dbSNP
rs1441532615	348	dbSNP
rs890993831	356	dbSNP
rs1437679758	360	dbSNP
rs1009359325	363	dbSNP
rs781326789	369	dbSNP
rs566076103	370	dbSNP
rs1005974810	371	dbSNP
rs1378451835	372	dbSNP
rs1053101	374	dbSNP
rs534973944	381	dbSNP
rs1227015424	385	dbSNP
rs1015839231	386	dbSNP
rs1245075321	391	dbSNP
rs958806445	392	dbSNP
rs780090320	393	dbSNP
rs1347982529	398	dbSNP
rs767942260	402	dbSNP
rs1459149558	403	dbSNP
rs1242479090	404	dbSNP
rs992021258	405	dbSNP
rs1268643281	422	dbSNP
rs992941145	424	dbSNP
rs917341627	425	dbSNP
rs950161283	427	dbSNP
rs1168108623	428	dbSNP
rs1401937834	428	dbSNP
rs1201473660	441	dbSNP
rs1024459170	444	dbSNP
rs1335468929	446	dbSNP
rs557937669	447	dbSNP
rs982959288	450	dbSNP
rs1286385444	454	dbSNP
rs980467321	455	dbSNP
rs908769593	463	dbSNP
rs1214284153	468	dbSNP
rs1188582267	472	dbSNP
rs1239267695	474	dbSNP
rs1468707646	476	dbSNP
rs578074446	477	dbSNP
rs926367062	478	dbSNP
rs553697397	486	dbSNP
rs17357180	488	dbSNP
rs1413279693	496	dbSNP
rs989583981	500	dbSNP
rs913814134	512	dbSNP
rs754907589	531	dbSNP
rs1174649179	538	dbSNP
rs1191185402	543	dbSNP
rs1425392282	557	dbSNP
rs1477927457	558	dbSNP
rs1402028660	561	dbSNP
rs1053107	578	dbSNP
rs899530091	579	dbSNP
rs1040886324	582	dbSNP
rs1336723959	583	dbSNP
rs1360211605	589	dbSNP
rs1333651895	592	dbSNP
rs900976890	596	dbSNP
rs1408635216	614	dbSNP
rs747880796	615	dbSNP
rs1274396594	618	dbSNP
rs771914639	620	dbSNP
rs1221679205	626	dbSNP
rs574064287	627	dbSNP
rs543072164	635	dbSNP
rs1214234204	640	dbSNP
rs1201222466	644	dbSNP
rs1265060498	645	dbSNP
rs1280955259	645	dbSNP
rs1436318093	647	dbSNP
rs768412748	648	dbSNP
rs1280523042	649	dbSNP
rs1362832090	649	dbSNP
rs1419592845	649	dbSNP
rs1491045342	649	dbSNP
rs200339046	649	dbSNP
rs3831951	649	dbSNP
rs57658162	649	dbSNP
rs747816308	649	dbSNP
rs932416959	650	dbSNP
rs1051247950	652	dbSNP
rs1016036309	654	dbSNP
rs1197947074	654	dbSNP
rs897400989	657	dbSNP
rs1352987231	661	dbSNP
rs1491288762	661	dbSNP
rs1491518075	662	dbSNP
rs1375922998	663	dbSNP
rs1288007496	668	dbSNP
rs890884533	668	dbSNP
rs1009326623	672	dbSNP
rs1020764264	673	dbSNP
rs1264856268	674	dbSNP
rs1198999913	675	dbSNP
rs903764839	675	dbSNP
rs772984666	677	dbSNP
rs1231809352	679	dbSNP
rs1183331008	683	dbSNP
rs1439682721	686	dbSNP
rs980682820	692	dbSNP
rs1000768226	702	dbSNP
rs749723930	703	dbSNP
rs1420723256	706	dbSNP
rs1162984195	711	dbSNP
rs559774801	717	dbSNP
rs1033009242	721	dbSNP
rs1308102672	725	dbSNP
rs768845539	731	dbSNP
rs958063929	740	dbSNP
rs958915959	743	dbSNP
rs1376276943	753	dbSNP
rs528758172	775	dbSNP
rs1444571991	788	dbSNP
rs1311412835	792	dbSNP
rs1354269248	795	dbSNP
rs1403006342	795	dbSNP
rs147539738	796	dbSNP
rs1210048562	818	dbSNP
rs1256964642	824	dbSNP
rs1456069307	828	dbSNP
rs1024780468	832	dbSNP
rs971487857	833	dbSNP
rs1331645324	834	dbSNP
rs1240593067	837	dbSNP
rs1440821691	840	dbSNP
rs982929716	842	dbSNP
rs1235088120	844	dbSNP
rs541282292	845	dbSNP
rs374401856	854	dbSNP
rs907711358	857	dbSNP
rs974768663	858	dbSNP
rs1377608903	860	dbSNP
rs1395376682	862	dbSNP
rs532951137	865	dbSNP
rs1310385530	880	dbSNP
rs1204791621	886	dbSNP
rs548000962	887	dbSNP
rs932466823	888	dbSNP
rs1484098911	889	dbSNP
rs1315625880	891	dbSNP
rs932276035	893	dbSNP
rs1050796662	896	dbSNP
rs17163112	898	dbSNP
rs1200400995	904	dbSNP
rs563270105	913	dbSNP
rs941473752	919	dbSNP
rs1425959053	921	dbSNP
rs78094079	923	dbSNP
rs945153828	932	dbSNP
rs1042192253	934	dbSNP
rs371742047	935	dbSNP
rs1423244345	937	dbSNP
rs1476947612	938	dbSNP
rs1201372749	940	dbSNP
rs140159347	947	dbSNP
rs1014213694	954	dbSNP
rs1465557274	956	dbSNP
rs61785869	957	dbSNP
rs111667462	958	dbSNP
rs895073371	962	dbSNP
rs1396409768	963	dbSNP
rs1013307081	970	dbSNP
rs1024335131	974	dbSNP
rs1220074957	979	dbSNP
rs1278372493	981	dbSNP
rs971458428	984	dbSNP
rs1311109434	989	dbSNP
rs1230385697	995	dbSNP
rs1254791810	998	dbSNP
rs1004333735	1005	dbSNP
rs1484514688	1008	dbSNP
rs1033617001	1012	dbSNP
rs1262918263	1025	dbSNP
rs10494394	1026	dbSNP
rs1011043229	1028	dbSNP
rs534988120	1029	dbSNP
rs974277636	1030	dbSNP
rs1173982046	1036	dbSNP
rs921486869	1042	dbSNP
rs922510342	1051	dbSNP
rs954123023	1053	dbSNP
rs1358408309	1073	dbSNP
rs985300618	1092	dbSNP
rs551756930	1093	dbSNP
rs941223927	1096	dbSNP
rs764604300	1098	dbSNP
rs986856033	1099	dbSNP
rs1366414618	1100	dbSNP
rs571825905	1108	dbSNP
rs1298555598	1115	dbSNP
rs1302730935	1126	dbSNP
rs774585267	1128	dbSNP
rs762162724	1129	dbSNP
rs1285858652	1132	dbSNP
rs1351963936	1143	dbSNP
rs1042161298	1147	dbSNP
rs1242938329	1153	dbSNP
rs537279736	1162	dbSNP
rs906104160	1163	dbSNP
rs1489088852	1176	dbSNP
rs555973200	1184	dbSNP
rs1157979380	1188	dbSNP
rs925039870	1191	dbSNP
rs767788666	1193	dbSNP
rs1260055712	1195	dbSNP
rs1378362822	1198	dbSNP
rs1464762986	1199	dbSNP
rs1159540418	1215	dbSNP
rs1421311174	1215	dbSNP
rs557509413	1217	dbSNP
rs936472108	1225	dbSNP
rs1433850506	1226	dbSNP
rs376006959	1232	dbSNP
rs1367708235	1235	dbSNP
rs1054982889	1248	dbSNP
rs1189649315	1255	dbSNP
rs1391583400	1264	dbSNP
rs895034964	1266	dbSNP
rs182324466	1272	dbSNP
rs1442416002	1273	dbSNP
rs187347421	1276	dbSNP
rs1020709189	1277	dbSNP
rs1163380923	1281	dbSNP
rs113975812	1282	dbSNP
rs1459368723	1283	dbSNP
rs569672944	1284	dbSNP
rs1236338773	1288	dbSNP
rs148860823	1288	dbSNP
rs1177640903	1295	dbSNP
rs953753118	1296	dbSNP
rs570669317	1301	dbSNP
rs1004217844	1302	dbSNP
rs1322314975	1304	dbSNP
rs1464664933	1305	dbSNP
rs1367535387	1307	dbSNP
rs1394572012	1308	dbSNP
rs1398791665	1313	dbSNP
rs557803615	1313	dbSNP
rs1444983204	1315	dbSNP
rs116651280	1316	dbSNP
rs962840123	1326	dbSNP
rs1322453818	1327	dbSNP
rs1378910377	1328	dbSNP
rs1226926076	1333	dbSNP
rs1247584378	1335	dbSNP
rs1315135841	1345	dbSNP
rs996056604	1347	dbSNP
rs962829132	1352	dbSNP
rs770627412	1356	dbSNP
rs1028515243	1362	dbSNP
rs754750260	1379	dbSNP
rs954429775	1380	dbSNP
rs1205155973	1383	dbSNP
rs987048489	1385	dbSNP
rs142561602	1387	dbSNP
rs1311752615	1391	dbSNP
rs1250299950	1393	dbSNP
rs563486248	1394	dbSNP
rs1477273448	1405	dbSNP
rs1167998003	1406	dbSNP
rs150991248	1408	dbSNP
rs79516637	1409	dbSNP
rs1330187145	1411	dbSNP
rs558944390	1413	dbSNP
rs528042378	1419	dbSNP
rs190218064	1423	dbSNP
rs764898340	1427	dbSNP
rs1446345976	1431	dbSNP
rs1424998079	1432	dbSNP
rs1315144425	1436	dbSNP
rs936456476	1439	dbSNP
rs1218235914	1442	dbSNP
rs1276478073	1443	dbSNP
rs867924638	1445	dbSNP
rs1320098040	1449	dbSNP
rs754871867	1450	dbSNP
rs991046969	1451	dbSNP
rs79909517	1452	dbSNP
rs949325353	1459	dbSNP
rs778903106	1460	dbSNP
rs1046307375	1461	dbSNP
rs140993675	1462	dbSNP
rs1171460445	1471	dbSNP
rs1425133282	1473	dbSNP
rs115114623	1474	dbSNP
rs368021271	1477	dbSNP
rs569230896	1477	dbSNP
rs1376461617	1478	dbSNP
rs748011894	1479	dbSNP
rs898557553	1481	dbSNP
rs13886	1486	dbSNP
rs1017254992	1487	dbSNP
rs962695371	1495	dbSNP
rs550903949	1496	dbSNP
rs1397811308	1506	dbSNP
rs1352721375	1512	dbSNP
rs972519393	1513	dbSNP
rs1264947336	1517	dbSNP
rs1025458135	1525	dbSNP
rs1197629345	1528	dbSNP
rs971216795	1530	dbSNP
rs1478693155	1534	dbSNP
rs553961307	1535	dbSNP
rs1349691354	1536	dbSNP
rs7414413	1539	dbSNP
rs772695080	1539	dbSNP
rs1028903777	1544	dbSNP
rs890002078	1549	dbSNP
rs927401923	1554	dbSNP
rs1401499314	1563	dbSNP
rs777606655	1565	dbSNP
rs1329430251	1570	dbSNP
rs1443108497	1576	dbSNP
rs567814738	1580	dbSNP
rs1304099685	1581	dbSNP
rs1229624403	1587	dbSNP
rs1270404296	1591	dbSNP
rs990207001	1593	dbSNP
rs1310866055	1595	dbSNP
rs536766960	1599	dbSNP
rs914791220	1600	dbSNP
rs746689765	1601	dbSNP
rs553471091	1611	dbSNP
rs1042005701	1614	dbSNP
rs566956642	1615	dbSNP
rs966948997	1618	dbSNP
rs1193657741	1622	dbSNP
rs1254229900	1625	dbSNP
rs977707687	1628	dbSNP
rs1179299966	1630	dbSNP
rs1032034217	1633	dbSNP
rs1365898518	1637	dbSNP
rs770567194	1648	dbSNP
rs1162893307	1650	dbSNP
rs538687060	1653	dbSNP
rs1463124863	1657	dbSNP
rs1236653933	1660	dbSNP
rs916319018	1666	dbSNP
rs576986152	1667	dbSNP
rs558910693	1668	dbSNP
rs981955439	1671	dbSNP
rs745318486	1672	dbSNP
rs907770209	1689	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.