ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uaGACGG-ACUC--GUAG-AGGAa 5' ||||| ||| ||| ||| Target 5' agCTGCCAGGAGGAAATCAGCCTt 3'	6 - 29	92.00	-16.50

Mutant ID	Mutant Position	Mutant Source
COSM9409298	19	COSMIC
COSM6811629	29	COSMIC
COSN511332	36	COSMIC
COSN18723740	57	COSMIC
COSN18742976	57	COSMIC
COSN1247479	65	COSMIC
COSN9125618	87	COSMIC
COSN8868581	116	COSMIC
COSN15016798	370	COSMIC
COSN26605148	718	COSMIC
COSN7089066	1136	COSMIC
COSN7089067	1355	COSMIC
COSN9125620	1946	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs373854935	1	dbSNP
rs1267028725	7	dbSNP
rs1357263071	19	dbSNP
rs769491446	21	dbSNP
rs775414005	26	dbSNP
rs763023511	27	dbSNP
rs1183518305	31	dbSNP
rs1260501394	41	dbSNP
rs1257493653	45	dbSNP
rs764099104	47	dbSNP
rs761440396	49	dbSNP
rs759022927	50	dbSNP
rs764688059	50	dbSNP
rs766982209	51	dbSNP
rs539731138	52	dbSNP
rs1166967262	54	dbSNP
rs765352958	55	dbSNP
rs551478067	56	dbSNP
rs553667704	57	dbSNP
rs752349075	57	dbSNP
rs781276229	58	dbSNP
rs1354782693	59	dbSNP
rs1394244253	61	dbSNP
rs746135823	71	dbSNP
rs1340852564	72	dbSNP
rs770025223	73	dbSNP
rs1272824776	75	dbSNP
rs1171149903	76	dbSNP
rs775473185	84	dbSNP
rs1004463376	87	dbSNP
rs1202630610	92	dbSNP
rs1343912034	93	dbSNP
rs1298035544	95	dbSNP
rs181933010	96	dbSNP
rs573809820	105	dbSNP
rs1448782747	110	dbSNP
rs1000963088	111	dbSNP
rs6058498	116	dbSNP
rs956644859	117	dbSNP
rs1431835981	119	dbSNP
rs556719578	125	dbSNP
rs1467513656	128	dbSNP
rs988066674	139	dbSNP
rs1416517367	142	dbSNP
rs1182977829	148	dbSNP
rs1389372829	159	dbSNP
rs923147303	160	dbSNP
rs1023002797	161	dbSNP
rs970062247	162	dbSNP
rs758403143	164	dbSNP
rs778429167	165	dbSNP
rs1251532239	166	dbSNP
rs1361721995	172	dbSNP
rs1319724660	180	dbSNP
rs978838025	181	dbSNP
rs1291884632	185	dbSNP
rs578152624	188	dbSNP
rs1228786763	191	dbSNP
rs1349848142	193	dbSNP
rs985967235	196	dbSNP
rs1351271224	197	dbSNP
rs1293174807	200	dbSNP
rs1456668606	206	dbSNP
rs752051268	207	dbSNP
rs1158637292	208	dbSNP
rs1413608913	220	dbSNP
rs1457158463	224	dbSNP
rs1410478322	236	dbSNP
rs141164955	239	dbSNP
rs1325174586	252	dbSNP
rs775593292	259	dbSNP
rs1250727418	261	dbSNP
rs1394883672	263	dbSNP
rs1038808646	268	dbSNP
rs1487323807	278	dbSNP
rs1388309438	290	dbSNP
rs1248217971	291	dbSNP
rs1327019477	294	dbSNP
rs919946097	300	dbSNP
rs545690357	312	dbSNP
rs781653191	326	dbSNP
rs912138301	327	dbSNP
rs944988028	335	dbSNP
rs896713349	339	dbSNP
rs1385280764	345	dbSNP
rs1332814997	356	dbSNP
rs563921905	358	dbSNP
rs976369387	369	dbSNP
rs920906729	370	dbSNP
rs1274380577	372	dbSNP
rs904981331	374	dbSNP
rs1000613623	393	dbSNP
rs1363839465	403	dbSNP
rs1174804354	404	dbSNP
rs1431080942	411	dbSNP
rs1426912384	421	dbSNP
rs1358843061	424	dbSNP
rs932332441	425	dbSNP
rs1464285493	427	dbSNP
rs1048110425	434	dbSNP
rs1246065761	451	dbSNP
rs1032474589	460	dbSNP
rs1194444597	470	dbSNP
rs892140051	475	dbSNP
rs888197418	479	dbSNP
rs572911536	480	dbSNP
rs1304455154	507	dbSNP
rs564361130	511	dbSNP
rs939868984	513	dbSNP
rs56398306	516	dbSNP
rs763083099	518	dbSNP
rs1323010015	519	dbSNP
rs1436432948	527	dbSNP
rs1037308392	529	dbSNP
rs1402191774	540	dbSNP
rs895724783	541	dbSNP
rs150714695	546	dbSNP
rs954590393	548	dbSNP
rs1022899480	549	dbSNP
rs905827332	550	dbSNP
rs746201207	552	dbSNP
rs1388056900	556	dbSNP
rs1017428024	563	dbSNP
rs1033093946	565	dbSNP
rs561739849	569	dbSNP
rs974186163	570	dbSNP
rs529002053	571	dbSNP
rs1459438251	574	dbSNP
rs988980871	579	dbSNP
rs1165924009	580	dbSNP
rs1281251452	586	dbSNP
rs1395563584	588	dbSNP
rs1340364683	592	dbSNP
rs1300252435	597	dbSNP
rs929991918	598	dbSNP
rs139000015	600	dbSNP
rs562745335	614	dbSNP
rs1393743770	615	dbSNP
rs749753143	616	dbSNP
rs1313000959	622	dbSNP
rs1294375901	627	dbSNP
rs1428684476	630	dbSNP
rs1045675421	632	dbSNP
rs1462312019	633	dbSNP
rs1342780373	634	dbSNP
rs533076633	635	dbSNP
rs577973822	636	dbSNP
rs974941926	639	dbSNP
rs1181623150	652	dbSNP
rs187570895	653	dbSNP
rs1251086186	655	dbSNP
rs1340549472	658	dbSNP
rs768415681	661	dbSNP
rs1276351131	664	dbSNP
rs922189869	667	dbSNP
rs1196689229	672	dbSNP
rs1484364931	673	dbSNP
rs936797043	675	dbSNP
rs932284836	676	dbSNP
rs1264165242	679	dbSNP
rs778428446	680	dbSNP
rs370873900	689	dbSNP
rs747701481	693	dbSNP
rs1256314757	694	dbSNP
rs1206009252	701	dbSNP
rs1279747139	702	dbSNP
rs1482533974	703	dbSNP
rs545678854	704	dbSNP
rs768531223	711	dbSNP
rs1428357181	712	dbSNP
rs1330495850	716	dbSNP
rs892193815	718	dbSNP
rs1387987351	727	dbSNP
rs1431069473	729	dbSNP
rs1036851454	731	dbSNP
rs868393651	733	dbSNP
rs1374338756	735	dbSNP
rs1459367257	746	dbSNP
rs1464426337	749	dbSNP
rs895631347	756	dbSNP
rs1009258891	761	dbSNP
rs1304587685	765	dbSNP
rs1029997450	766	dbSNP
rs1381489643	768	dbSNP
rs1254188245	776	dbSNP
rs190649769	781	dbSNP
rs774430370	782	dbSNP
rs1354456574	789	dbSNP
rs949869367	791	dbSNP
rs1229437253	792	dbSNP
rs1302689226	794	dbSNP
rs1280498072	795	dbSNP
rs142685502	797	dbSNP
rs1212854639	799	dbSNP
rs772772052	800	dbSNP
rs1485731753	807	dbSNP
rs1227186907	809	dbSNP
rs573163089	814	dbSNP
rs182153872	815	dbSNP
rs1239284845	816	dbSNP
rs1017899460	817	dbSNP
rs963260045	828	dbSNP
rs973615548	848	dbSNP
rs1395051937	849	dbSNP
rs1027055402	850	dbSNP
rs1455673534	853	dbSNP
rs1301780435	857	dbSNP
rs1410212241	861	dbSNP
rs773155838	863	dbSNP
rs1345273615	876	dbSNP
rs1477815650	884	dbSNP
rs951449420	887	dbSNP
rs1201380840	901	dbSNP
rs1209768442	901	dbSNP
rs556469821	902	dbSNP
rs1247901010	905	dbSNP
rs1000199095	908	dbSNP
rs760566040	911	dbSNP
rs770825691	914	dbSNP
rs1010708937	917	dbSNP
rs1019004782	918	dbSNP
rs949380040	923	dbSNP
rs1223919932	924	dbSNP
rs1343763489	927	dbSNP
rs1272730956	930	dbSNP
rs966143022	931	dbSNP
rs974889381	934	dbSNP
rs578157339	936	dbSNP
rs981050543	937	dbSNP
rs1346954351	947	dbSNP
rs926846383	948	dbSNP
rs1029190659	952	dbSNP
rs1400270351	953	dbSNP
rs1175786324	955	dbSNP
rs936869829	957	dbSNP
rs1389915939	958	dbSNP
rs952436289	965	dbSNP
rs1187127912	981	dbSNP
rs1190271789	983	dbSNP
rs1489722764	985	dbSNP
rs540566222	987	dbSNP
rs538941101	990	dbSNP
rs961082566	1002	dbSNP
rs1276018268	1003	dbSNP
rs972439815	1006	dbSNP
rs1379406366	1012	dbSNP
rs945096943	1014	dbSNP
rs759236680	1015	dbSNP
rs1051476366	1017	dbSNP
rs1390010090	1017	dbSNP
rs554413249	1018	dbSNP
rs1270713555	1020	dbSNP
rs890127429	1024	dbSNP
rs949858064	1036	dbSNP
rs1314892367	1042	dbSNP
rs1007613130	1045	dbSNP
rs1038963621	1054	dbSNP
rs1167026322	1055	dbSNP
rs899064183	1063	dbSNP
rs1181820102	1066	dbSNP
rs1420784248	1066	dbSNP
rs994703692	1068	dbSNP
rs1044293558	1069	dbSNP
rs927178038	1074	dbSNP
rs1485792667	1079	dbSNP
rs1313384862	1081	dbSNP
rs774822348	1082	dbSNP
rs186753664	1088	dbSNP
rs1273587331	1093	dbSNP
rs540190693	1099	dbSNP
rs1465283946	1102	dbSNP
rs1354229437	1108	dbSNP
rs1295195351	1118	dbSNP
rs1415476669	1122	dbSNP
rs1350039862	1123	dbSNP
rs765561776	1128	dbSNP
rs561677063	1130	dbSNP
rs1040402026	1132	dbSNP
rs34764382	1141	dbSNP
rs970773012	1141	dbSNP
rs981153490	1155	dbSNP
rs867311963	1180	dbSNP
rs1355446962	1182	dbSNP
rs1210571317	1186	dbSNP
rs1404083032	1188	dbSNP
rs1420741160	1194	dbSNP
rs926926209	1197	dbSNP
rs901969196	1199	dbSNP
rs1250876156	1214	dbSNP
rs762700620	1215	dbSNP
rs1029139596	1216	dbSNP
rs768366400	1218	dbSNP
rs6061045	1220	dbSNP
rs914079361	1221	dbSNP
rs763102638	1234	dbSNP
rs1233920302	1243	dbSNP
rs1328619301	1256	dbSNP
rs1015181452	1262	dbSNP
rs190095953	1275	dbSNP
rs1051527493	1277	dbSNP
rs911600710	1283	dbSNP
rs1293304190	1286	dbSNP
rs1434770221	1287	dbSNP
rs764217371	1289	dbSNP
rs145090295	1292	dbSNP
rs1397255977	1298	dbSNP
rs972429728	1299	dbSNP
rs528916470	1305	dbSNP
rs1461475012	1313	dbSNP
rs11697022	1322	dbSNP
rs1410549229	1324	dbSNP
rs943078374	1337	dbSNP
rs762266024	1338	dbSNP
rs1408267142	1342	dbSNP
rs1039090867	1344	dbSNP
rs1452363769	1363	dbSNP
rs899121884	1367	dbSNP
rs994705697	1372	dbSNP
rs1464426521	1373	dbSNP
rs1047605554	1375	dbSNP
rs1218122208	1377	dbSNP
rs1412853273	1378	dbSNP
rs866760743	1380	dbSNP
rs1459255097	1390	dbSNP
rs1217787117	1398	dbSNP
rs886276902	1401	dbSNP
rs1304684952	1405	dbSNP
rs917002046	1406	dbSNP
rs1163435214	1408	dbSNP
rs1387173934	1417	dbSNP
rs1286539140	1421	dbSNP
rs182558347	1433	dbSNP
rs188187497	1436	dbSNP
rs979920247	1439	dbSNP
rs1396637809	1440	dbSNP
rs533189360	1440	dbSNP
rs1293689869	1441	dbSNP
rs935868045	1443	dbSNP
rs1259581743	1447	dbSNP
rs1054741350	1455	dbSNP
rs1255590955	1459	dbSNP
rs913154845	1460	dbSNP
rs1208965226	1465	dbSNP
rs551300380	1468	dbSNP
rs193175077	1471	dbSNP
rs989732308	1472	dbSNP
rs185418189	1475	dbSNP
rs966880799	1477	dbSNP
rs996243241	1477	dbSNP
rs1301804002	1479	dbSNP
rs1361997456	1479	dbSNP
rs987623978	1483	dbSNP
rs911668180	1486	dbSNP
rs188983745	1490	dbSNP
rs1173960859	1493	dbSNP
rs1428887640	1497	dbSNP
rs1371221935	1498	dbSNP
rs1271221160	1500	dbSNP
rs1333796061	1504	dbSNP
rs750923667	1505	dbSNP
rs1248300486	1506	dbSNP
rs1255535970	1508	dbSNP
rs376361287	1509	dbSNP
rs1185977685	1510	dbSNP
rs1486735953	1520	dbSNP
rs920264261	1524	dbSNP
rs887917125	1527	dbSNP
rs1006399818	1538	dbSNP
rs930273531	1541	dbSNP
rs1337887793	1545	dbSNP
rs1015585958	1547	dbSNP
rs1047657748	1551	dbSNP
rs750870356	1552	dbSNP
rs1228765339	1556	dbSNP
rs962363782	1564	dbSNP
rs1479767289	1573	dbSNP
rs1176877233	1574	dbSNP
rs1292127188	1577	dbSNP
rs567361701	1580	dbSNP
rs1252575121	1589	dbSNP
rs1408952548	1595	dbSNP
rs531688870	1604	dbSNP
rs1169483042	1611	dbSNP
rs550276737	1618	dbSNP
rs1367819956	1619	dbSNP
rs192600038	1621	dbSNP
rs1476239293	1623	dbSNP
rs114350216	1625	dbSNP
rs1181690988	1626	dbSNP
rs906650012	1630	dbSNP
rs554077096	1637	dbSNP
rs1002704865	1640	dbSNP
rs1024006947	1649	dbSNP
rs566337942	1661	dbSNP
rs114986928	1664	dbSNP
rs1460125227	1665	dbSNP
rs1230900036	1669	dbSNP
rs1336357081	1673	dbSNP
rs1293405422	1677	dbSNP
rs1237816243	1684	dbSNP
rs555400395	1685	dbSNP
rs1010881669	1692	dbSNP
rs1021245572	1698	dbSNP
rs1350517341	1704	dbSNP
rs1462978929	1710	dbSNP
rs573711198	1710	dbSNP
rs1158539362	1711	dbSNP
rs1395249737	1711	dbSNP
rs1468737788	1712	dbSNP
rs1167985892	1720	dbSNP
rs1182964282	1725	dbSNP
rs1034105567	1727	dbSNP
rs34089906	1730	dbSNP
rs1251643071	1737	dbSNP
rs543959797	1742	dbSNP
rs556187571	1744	dbSNP
rs1469320824	1748	dbSNP
rs1228868437	1752	dbSNP
rs1273200429	1768	dbSNP
rs1348088178	1769	dbSNP
rs761042903	1769	dbSNP
rs756370085	1770	dbSNP
rs1239468488	1771	dbSNP
rs1305578449	1775	dbSNP
rs1301011385	1776	dbSNP
rs974901521	1779	dbSNP
rs1204030574	1784	dbSNP
rs913136466	1787	dbSNP
rs992774802	1790	dbSNP
rs1395132926	1799	dbSNP
rs778645409	1807	dbSNP
rs945989141	1808	dbSNP
rs976040745	1815	dbSNP
rs923244186	1816	dbSNP
rs932000128	1819	dbSNP
rs747706478	1820	dbSNP
rs983067930	1821	dbSNP
rs766508039	1826	dbSNP
rs1195907802	1836	dbSNP
rs1455032052	1862	dbSNP
rs577683457	1865	dbSNP
rs370747219	1866	dbSNP
rs907823301	1869	dbSNP
rs1285787923	1874	dbSNP
rs949970187	1879	dbSNP
rs6061046	1886	dbSNP
rs777306936	1888	dbSNP
rs139924671	1889	dbSNP
rs746502425	1890	dbSNP
rs1236304808	1905	dbSNP
rs779091944	1918	dbSNP
rs1167509868	1921	dbSNP
rs893882026	1921	dbSNP
rs1319087772	1925	dbSNP
rs6121290	1930	dbSNP
rs770911462	1942	dbSNP
rs1010934115	1943	dbSNP
rs527570770	1949	dbSNP
rs1431310391	1957	dbSNP
rs1310519174	1970	dbSNP
rs542559175	1972	dbSNP
rs1358111492	1975	dbSNP
rs1020964287	1976	dbSNP
rs1166627254	1977	dbSNP
rs560925639	1979	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.