>NPAT|NM_002519|3'UTR
   1 ACATTCTGGACACATAAGAACAGTAGGTGTTAAAAACTCATATCCCTTAAACTGAGTGTAGGGAATGGGATATTGACAGA
  81 ATCTGAAAGCATGACCTGCACTTTCATTGTACTGAAACTTCACTTTATATCTAAATCATGCTGTTTCTGAAACAGCTTCC
 161 TAGTTTGTAAATAGACTTACTTGGTATATTTTTATTTTGGGAAAAACGTTTGCAGAAATGTAAGTAAAGCCAATCTGCAA
 241 AACTGTATAGCTTTGACAATTCCATATTGTAAATACTGTGTAAATCTTGTTGAAATAGAGGTTAAATCAACCTAATGTTC
 321 TTACACTGTGTTTTTTGACTTCTTATGATCCCTAATTCTGAGACTTACTCATCTGGAATAGTTTCTCACTTGTTTTGGAG
 401 GAAAATGATGCTTATTCTTATAAATTACCTTCAGTAACTATTCAATAAGACATTTAAATACACAATCACTAAGTACTACA
 481 AAATAATAACCACCTCAACTGATAGCAAAATATCTAATTAGAAATCAACAGTTTGATGAGTTTTTTTCCTAGAGTGAATA
 561 CTACCCCTTTCTTATAATTGCTGTAAGTATTATATTTCTGGAATTTCACACTTTACCTACTCTGATCACTCTGTTCTCTT
 641 TGTTTTAAAGAGAGAATTTTGTAAACCATTTATTGAATGTTCTGATCTTTCATTTATAACTTAAGATTTTTTACAGAACT
 721 TTAAGTGGTTAGACTATTGAGCCATAAAATTTTGGTTTGTAGTAGAGATTTCTCGGTATGTACTTTTCCCCTAAATGCTG
 801 GGGATTTAAAGAATTGCACTAATTCATAGAACTCTTTACATTGTAGTGACCAATGCACATATAATTTAAATCTTGTGTTC
 881 TATGTGAGAGTTTATGTGGAGGATTATCTTCTGGTGTTGTGTCCATCATAAGATGTAAAAAGAGAAATCATAGTTTTATA
 961 TTTTAGTTTCTCCTACAATGGGTATGTTAAATAACTATATCTATACTTAGATATAGTGCATTGTCTGTGTCCCTTAAAGT
1041 GTTACAAAGAGTTTCTCTCAAAAGGTCCTTAAGGAGAGTGAGAGATGAGAGAGGTGCCTTCTCTGTATTAGACTCACACA
1121 CATCAGTCCAGTTGAAGAATTGGTTTAAACTTTTAAATGAGAACAGAAATTATAATTAGTACCTGACCAATTGTGCCTAG
1201 ATATTAAAGTATGTTCTCTTTAGTACTTTCCGTTCAATTAAATATCAGTATATTAGGGATTTTTTTCTACAGGTGTATAT
1281 TTTGTTGAAGAGTCACTTTCCTCAACCATTTTTTTGTACTGGGCCTTTAAAAAAATAAATCCATCCAACTTTAACACAAC
1361 ATTTCTCAGTGTAGAAATCATGTCTTCTTAATTGCTGAACCTTACTGCAAAAACTTGTGATGTAAGAAATTTGTATGGTG
1441 TGGCAGTGGTCTATTCCTAAGGAACTAAATATCATATAGTTAATGTTTATTTAACTCAGCTTGAGACTGTACTACAGTTA
1521 GGTTTGAATAAATATTTTCATTAACTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uacauccGGA--AAUCUAGUGAa 5' ||| | ||||||| Target 5' cactttaCCTACTCTGATCACTc 3'	609 - 631	144.00	-10.70
2	miRNA 3' uaCAUCCGGA--AA--UCU-AGUGAa 5' ||| ::| || ||| ||||| Target 5' gtGTATATTTTGTTGAAGAGTCACTt 3'	1273 - 1298	119.00	-7.80
3	miRNA 3' uacAUCCGGAA---AUCU-AGUGAa 5' ||| :: | |||| |:||| Target 5' tccTAGTTTGTAAATAGACTTACTt 3'	158 - 182	106.00	-7.80

Mutant ID	Mutant Position	Mutant Source
COSN30124006	17	COSMIC
COSN31597432	36	COSMIC
COSN30518910	69	COSMIC
COSN31550621	96	COSMIC
COSN24293426	105	COSMIC
COSN31574167	163	COSMIC
COSN21630211	219	COSMIC
COSN26551086	368	COSMIC
COSN10077490	373	COSMIC
COSN5893918	533	COSMIC
COSN31486967	552	COSMIC
COSN20108714	558	COSMIC
COSN21400677	631	COSMIC
COSN28444209	637	COSMIC
COSN28796159	683	COSMIC
COSN31518577	858	COSMIC
COSN1512754	905	COSMIC
COSN31491234	1010	COSMIC
COSN14537810	1016	COSMIC
COSN31607847	1083	COSMIC
COSN16631681	1115	COSMIC
COSN30212589	1332	COSMIC
COSN6406072	1427	COSMIC
COSN26129717	1563	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1248868442	6	dbSNP
rs56219135	14	dbSNP
rs1324688987	15	dbSNP
rs544760764	21	dbSNP
rs1222218124	22	dbSNP
rs1322994773	25	dbSNP
rs1280151876	26	dbSNP
rs774093155	27	dbSNP
rs1334167457	32	dbSNP
rs1265132672	35	dbSNP
rs780057547	37	dbSNP
rs765934115	41	dbSNP
rs1406504421	46	dbSNP
rs140738694	49	dbSNP
rs1396467983	60	dbSNP
rs1461534262	63	dbSNP
rs562482906	68	dbSNP
rs1434029369	79	dbSNP
rs1273962885	88	dbSNP
rs1362591111	104	dbSNP
rs1016349965	109	dbSNP
rs1275792780	123	dbSNP
rs544086322	129	dbSNP
rs1213984210	134	dbSNP
rs1014328082	137	dbSNP
rs950444676	139	dbSNP
rs1398878574	143	dbSNP
rs767559401	154	dbSNP
rs1242998973	157	dbSNP
rs1476686315	168	dbSNP
rs576388408	172	dbSNP
rs1423055227	174	dbSNP
rs376309624	181	dbSNP
rs554923299	184	dbSNP
rs1411908692	187	dbSNP
rs1404931259	198	dbSNP
rs1322680020	200	dbSNP
rs763373380	206	dbSNP
rs554153206	207	dbSNP
rs1296245306	208	dbSNP
rs1039356976	213	dbSNP
rs1007442699	225	dbSNP
rs1233358620	230	dbSNP
rs375994359	233	dbSNP
rs1476884448	238	dbSNP
rs888720917	247	dbSNP
rs1227335876	251	dbSNP
rs1248404650	256	dbSNP
rs35895314	264	dbSNP
rs1194153474	276	dbSNP
rs536361496	277	dbSNP
rs930191276	285	dbSNP
rs1197061041	294	dbSNP
rs913305859	301	dbSNP
rs1053217007	308	dbSNP
rs938677489	327	dbSNP
rs572461582	337	dbSNP
rs980515751	340	dbSNP
rs1156849637	345	dbSNP
rs1365883244	345	dbSNP
rs1041225239	346	dbSNP
rs948956278	347	dbSNP
rs942472227	348	dbSNP
rs1320760514	352	dbSNP
rs1338149940	353	dbSNP
rs910922093	365	dbSNP
rs984913715	385	dbSNP
rs1442252352	395	dbSNP
rs989179620	397	dbSNP
rs554409820	399	dbSNP
rs1285690045	407	dbSNP
rs372789363	410	dbSNP
rs762060580	421	dbSNP
rs1443657735	426	dbSNP
rs1441896244	427	dbSNP
rs1026019390	428	dbSNP
rs1234813445	436	dbSNP
rs1437989848	445	dbSNP
rs923613161	446	dbSNP
rs1379400873	447	dbSNP
rs1435298129	452	dbSNP
rs1354765413	453	dbSNP
rs966195639	456	dbSNP
rs1017231072	459	dbSNP
rs1329202981	461	dbSNP
rs1378677607	461	dbSNP
rs542305729	465	dbSNP
rs1309031770	474	dbSNP
rs903742494	486	dbSNP
rs1412587157	498	dbSNP
rs1236134050	499	dbSNP
rs34666544	504	dbSNP
rs1022250073	508	dbSNP
rs1426982663	511	dbSNP
rs571857473	514	dbSNP
rs1344487046	515	dbSNP
rs1009384881	519	dbSNP
rs970612529	536	dbSNP
rs892342610	537	dbSNP
rs1053604019	538	dbSNP
rs1439002405	539	dbSNP
rs1386886249	545	dbSNP
rs1024772898	547	dbSNP
rs1198200576	548	dbSNP
rs1011997241	551	dbSNP
rs958838055	553	dbSNP
rs1454531222	554	dbSNP
rs1200981936	559	dbSNP
rs397967218	560	dbSNP
rs61380955	560	dbSNP
rs1034820855	575	dbSNP
rs1005619324	576	dbSNP
rs1330244973	580	dbSNP
rs1395753150	590	dbSNP
rs550527227	593	dbSNP
rs1299562237	610	dbSNP
rs1331047064	611	dbSNP
rs1044377279	612	dbSNP
rs1280206548	614	dbSNP
rs888466619	620	dbSNP
rs1218301031	622	dbSNP
rs1031241215	642	dbSNP
rs948761545	647	dbSNP
rs538506674	668	dbSNP
rs1233702002	672	dbSNP
rs1468798257	675	dbSNP
rs1211415802	677	dbSNP
rs1209639423	679	dbSNP
rs909452296	680	dbSNP
rs75150151	685	dbSNP
rs1425214551	690	dbSNP
rs1476547838	691	dbSNP
rs1427430960	696	dbSNP
rs1169603717	697	dbSNP
rs1418130836	703	dbSNP
rs1459070145	705	dbSNP
rs994454360	712	dbSNP
rs1292167846	713	dbSNP
rs1390414527	722	dbSNP
rs1383309045	729	dbSNP
rs548924464	734	dbSNP
rs115478376	736	dbSNP
rs1338590741	743	dbSNP
rs1041236545	744	dbSNP
rs1218082141	753	dbSNP
rs919394250	772	dbSNP
rs559864050	773	dbSNP
rs551055619	774	dbSNP
rs768943057	775	dbSNP
rs976660020	776	dbSNP
rs1217328728	779	dbSNP
rs1265242866	785	dbSNP
rs575292074	788	dbSNP
rs942838710	789	dbSNP
rs1487229913	797	dbSNP
rs749048506	804	dbSNP
rs182764040	808	dbSNP
rs1257545251	814	dbSNP
rs965800465	821	dbSNP
rs1017784956	825	dbSNP
rs557507721	830	dbSNP
rs200169344	838	dbSNP
rs967728767	839	dbSNP
rs1022217508	841	dbSNP
rs1313597413	842	dbSNP
rs1301947261	843	dbSNP
rs1394562746	844	dbSNP
rs1053970021	853	dbSNP
rs1377118071	874	dbSNP
rs1339726163	877	dbSNP
rs936432089	882	dbSNP
rs1313267060	884	dbSNP
rs544202100	894	dbSNP
rs573846485	896	dbSNP
rs1256114998	920	dbSNP
rs1483933934	924	dbSNP
rs1161154690	929	dbSNP
rs561134444	933	dbSNP
rs189818176	935	dbSNP
rs373119424	936	dbSNP
rs187939321	939	dbSNP
rs1419670188	941	dbSNP
rs1174793070	947	dbSNP
rs1049059393	950	dbSNP
rs769843108	953	dbSNP
rs929522389	956	dbSNP
rs1482395237	959	dbSNP
rs959498927	968	dbSNP
rs1040519206	970	dbSNP
rs1311225429	972	dbSNP
rs147236990	978	dbSNP
rs1259787554	982	dbSNP
rs539117126	989	dbSNP
rs1016078361	997	dbSNP
rs3758910	999	dbSNP
rs556765145	1003	dbSNP
rs1313505744	1006	dbSNP
rs1207414552	1007	dbSNP
rs1025657733	1011	dbSNP
rs1254843783	1023	dbSNP
rs1457949267	1028	dbSNP
rs1241779756	1029	dbSNP
rs1200109734	1045	dbSNP
rs1236788303	1048	dbSNP
rs1439461500	1054	dbSNP
rs1192356656	1059	dbSNP
rs1427873020	1064	dbSNP
rs1431885584	1067	dbSNP
rs967696205	1081	dbSNP
rs1355149781	1082	dbSNP
rs545627735	1087	dbSNP
rs143809771	1088	dbSNP
rs183226283	1091	dbSNP
rs1226053908	1094	dbSNP
rs901442913	1096	dbSNP
rs1397917796	1105	dbSNP
rs1445768001	1110	dbSNP
rs1293827570	1113	dbSNP
rs564556761	1116	dbSNP
rs555784535	1119	dbSNP
rs1217355565	1132	dbSNP
rs1007123841	1140	dbSNP
rs769518236	1140	dbSNP
rs546254662	1146	dbSNP
rs956583651	1147	dbSNP
rs1317976413	1166	dbSNP
rs1035379833	1183	dbSNP
rs889991397	1184	dbSNP
rs533827918	1186	dbSNP
rs1456994344	1190	dbSNP
rs969668516	1199	dbSNP
rs1248771989	1202	dbSNP
rs1487811319	1204	dbSNP
rs1191093876	1211	dbSNP
rs566116234	1218	dbSNP
rs936424743	1221	dbSNP
rs1424483381	1231	dbSNP
rs1478835755	1232	dbSNP
rs902230996	1235	dbSNP
rs1423182759	1242	dbSNP
rs1406107479	1244	dbSNP
rs1042053493	1249	dbSNP
rs190734772	1250	dbSNP
rs1012921621	1252	dbSNP
rs1351362412	1267	dbSNP
rs1434281825	1267	dbSNP
rs532836614	1267	dbSNP
rs949025402	1269	dbSNP
rs1340672677	1273	dbSNP
rs1290606854	1275	dbSNP
rs917567661	1277	dbSNP
rs1454232956	1284	dbSNP
rs1401317047	1288	dbSNP
rs568729031	1295	dbSNP
rs756767954	1299	dbSNP
rs991170015	1305	dbSNP
rs937942863	1307	dbSNP
rs909074605	1319	dbSNP
rs1420320118	1323	dbSNP
rs1188872815	1324	dbSNP
rs1320265894	1334	dbSNP
rs1049028491	1338	dbSNP
rs576627964	1345	dbSNP
rs186904401	1349	dbSNP
rs1489518824	1358	dbSNP
rs1207478974	1359	dbSNP
rs1262500611	1359	dbSNP
rs952776355	1360	dbSNP
rs1486483214	1361	dbSNP
rs1466117593	1363	dbSNP
rs1415769935	1364	dbSNP
rs1025711861	1365	dbSNP
rs1265314662	1367	dbSNP
rs1218772468	1370	dbSNP
rs898063736	1371	dbSNP
rs1041050043	1377	dbSNP
rs944889385	1380	dbSNP
rs181715087	1388	dbSNP
rs1339241725	1390	dbSNP
rs1448366657	1396	dbSNP
rs1285575211	1405	dbSNP
rs1377520811	1408	dbSNP
rs562062404	1410	dbSNP
rs972742112	1443	dbSNP
rs556838406	1445	dbSNP
rs910743809	1453	dbSNP
rs1217614545	1460	dbSNP
rs1287112486	1462	dbSNP
rs965399909	1469	dbSNP
rs1019623651	1474	dbSNP
rs946408827	1475	dbSNP
rs915010190	1476	dbSNP
rs1443063182	1479	dbSNP
rs987775769	1481	dbSNP
rs56882203	1495	dbSNP
rs956599488	1499	dbSNP
rs540147269	1509	dbSNP
rs1366832317	1524	dbSNP
rs890008697	1526	dbSNP
rs1182781633	1539	dbSNP
rs927902632	1542	dbSNP
rs1245959429	1549	dbSNP
rs1421484248	1552	dbSNP
rs1156748947	1554	dbSNP
rs1291664058	1568	dbSNP
rs1032924798	1576	dbSNP
rs1383687798	1578	dbSNP
rs1414538759	1580	dbSNP
rs1001015849	1585	dbSNP
rs1335541383	1585	dbSNP
rs1310482531	1593	dbSNP
rs777052526	1593	dbSNP
rs902282384	1595	dbSNP
rs1042146659	1606	dbSNP
rs116258775	1613	dbSNP
rs1465295495	1623	dbSNP
rs1309169220	1626	dbSNP
rs896198621	1633	dbSNP
rs952102476	1635	dbSNP
rs1027724672	1636	dbSNP
rs560484247	1650	dbSNP
rs1429436514	1651	dbSNP
rs536574329	1661	dbSNP
rs993440976	1662	dbSNP
rs897860549	1663	dbSNP
rs1363161222	1667	dbSNP
rs1478281654	1671	dbSNP
rs1173838963	1676	dbSNP
rs1040621015	1677	dbSNP
rs1331579743	1705	dbSNP
rs1054793374	1706	dbSNP
rs1267309016	1707	dbSNP
rs1445036862	1708	dbSNP
rs545538016	1709	dbSNP
rs181607205	1714	dbSNP
rs1192278170	1718	dbSNP
rs1469459942	1721	dbSNP
rs1273252879	1722	dbSNP
rs1332204525	1722	dbSNP
rs369162910	1722	dbSNP
rs937935224	1726	dbSNP
rs1197692173	1727	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.