ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cuuccuccGUCGCCACUCUAGu 5' :| | ||||||| Target 5' ttatataaTACCTTTGAGATCt 3'	458 - 479	142.00	-7.20
2	miRNA 3' cuUCCUCCG-----UCGC-CACUCUAgu 5' | | ||| || | |||||| Target 5' tcAAGTGGCCTTGAAGAGACTGAGATaa 3'	23 - 50	124.00	-9.10
3	miRNA 3' cuUCCUCCGUC-GCCACUCUAgu 5' ||||||:|| | |||| | Target 5' ctAGGAGGTAGTCACTGAGCTgg 3'	1380 - 1402	120.00	-17.86

Mutant ID	Mutant Position	Mutant Source
COSN30167965	58	COSMIC
COSN31525211	58	COSMIC
COSN30147661	85	COSMIC
COSN30171398	118	COSMIC
COSN30533918	131	COSMIC
COSN20581342	143	COSMIC
COSN28118038	600	COSMIC
COSN24110062	985	COSMIC
COSN27665348	1547	COSMIC
COSN20102981	1573	COSMIC
COSN24577129	1682	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1054182579	4	dbSNP
rs375229400	11	dbSNP
rs1346453674	14	dbSNP
rs369240120	15	dbSNP
rs1319449703	17	dbSNP
rs1362821346	21	dbSNP
rs1022343800	22	dbSNP
rs1247852409	30	dbSNP
rs1438317454	31	dbSNP
rs1315655241	32	dbSNP
rs750764094	38	dbSNP
rs1461068278	39	dbSNP
rs1458771443	40	dbSNP
rs1389669080	41	dbSNP
rs1161334377	43	dbSNP
rs1399050290	47	dbSNP
rs1178864590	48	dbSNP
rs1226526852	49	dbSNP
rs192633113	52	dbSNP
rs780842240	53	dbSNP
rs761355963	54	dbSNP
rs1193419205	55	dbSNP
rs769695159	66	dbSNP
rs1465015785	75	dbSNP
rs1010096802	80	dbSNP
rs996893954	86	dbSNP
rs183348294	89	dbSNP
rs901804025	90	dbSNP
rs114829203	107	dbSNP
rs1234462525	115	dbSNP
rs1279201142	128	dbSNP
rs1028287361	135	dbSNP
rs983450410	166	dbSNP
rs137857003	167	dbSNP
rs962875349	170	dbSNP
rs1006066336	173	dbSNP
rs531919131	176	dbSNP
rs1323056106	177	dbSNP
rs1409580826	180	dbSNP
rs1290905448	197	dbSNP
rs771551538	201	dbSNP
rs1344756606	214	dbSNP
rs1177411085	217	dbSNP
rs1437059884	224	dbSNP
rs1393812933	226	dbSNP
rs1173359270	236	dbSNP
rs1450499656	239	dbSNP
rs961746386	240	dbSNP
rs766442081	246	dbSNP
rs972958769	254	dbSNP
rs917419267	257	dbSNP
rs947792688	274	dbSNP
rs1345595512	280	dbSNP
rs980487413	283	dbSNP
rs927607274	284	dbSNP
rs550527544	289	dbSNP
rs1309083750	292	dbSNP
rs939003412	292	dbSNP
rs187647057	295	dbSNP
rs926959680	301	dbSNP
rs532825148	304	dbSNP
rs1287005783	309	dbSNP
rs751501741	314	dbSNP
rs935659473	337	dbSNP
rs1347890773	340	dbSNP
rs760847471	354	dbSNP
rs1054513217	359	dbSNP
rs1312857745	378	dbSNP
rs1430768759	378	dbSNP
rs1375208664	402	dbSNP
rs1167862386	403	dbSNP
rs764056548	404	dbSNP
rs1427489163	405	dbSNP
rs1414109827	410	dbSNP
rs1189389435	427	dbSNP
rs913011824	431	dbSNP
rs1274382345	433	dbSNP
rs551397884	443	dbSNP
rs946586288	447	dbSNP
rs1342962053	450	dbSNP
rs1043538829	467	dbSNP
rs754157972	468	dbSNP
rs945780352	473	dbSNP
rs566300581	478	dbSNP
rs1307093368	479	dbSNP
rs745824315	481	dbSNP
rs997324192	482	dbSNP
rs1353684533	493	dbSNP
rs578124123	504	dbSNP
rs192343438	521	dbSNP
rs996059547	528	dbSNP
rs1411034847	529	dbSNP
rs1399268485	531	dbSNP
rs184288281	540	dbSNP
rs1204131046	546	dbSNP
rs1004178549	547	dbSNP
rs887677219	548	dbSNP
rs540059519	552	dbSNP
rs1015864298	559	dbSNP
rs1473939384	565	dbSNP
rs34498316	574	dbSNP
rs962951176	593	dbSNP
rs1199449280	617	dbSNP
rs1005580001	624	dbSNP
rs1248364892	642	dbSNP
rs1023121966	648	dbSNP
rs1488710386	649	dbSNP
rs1274009177	651	dbSNP
rs969194769	654	dbSNP
rs189760159	663	dbSNP
rs1292684243	671	dbSNP
rs961522658	677	dbSNP
rs994620950	683	dbSNP
rs1169294214	689	dbSNP
rs1390329268	693	dbSNP
rs1429540200	694	dbSNP
rs1372271425	695	dbSNP
rs1327491806	698	dbSNP
rs1024487489	705	dbSNP
rs1306622402	706	dbSNP
rs1159204556	708	dbSNP
rs1454911807	711	dbSNP
rs1181047053	720	dbSNP
rs1361970129	720	dbSNP
rs987759036	721	dbSNP
rs1367819254	732	dbSNP
rs7756421	743	dbSNP
rs946615724	748	dbSNP
rs1467280993	753	dbSNP
rs1272813632	757	dbSNP
rs1329432657	759	dbSNP
rs1229621795	772	dbSNP
rs1043566995	777	dbSNP
rs921107500	779	dbSNP
rs1239037396	784	dbSNP
rs1372384103	796	dbSNP
rs1306001213	808	dbSNP
rs1439197853	815	dbSNP
rs980369056	817	dbSNP
rs1382226134	818	dbSNP
rs1157915221	831	dbSNP
rs1341661020	834	dbSNP
rs555913734	840	dbSNP
rs1171472183	842	dbSNP
rs1396817465	842	dbSNP
rs957846488	855	dbSNP
rs1252578428	861	dbSNP
rs1194249315	863	dbSNP
rs1004250491	866	dbSNP
rs1037032155	869	dbSNP
rs1204638328	874	dbSNP
rs898774450	875	dbSNP
rs1283111974	898	dbSNP
rs1489337756	907	dbSNP
rs779861311	912	dbSNP
rs1270402624	915	dbSNP
rs1266510810	917	dbSNP
rs1229228981	947	dbSNP
rs1324333448	955	dbSNP
rs561271006	958	dbSNP
rs1414894512	967	dbSNP
rs574056881	968	dbSNP
rs530376659	969	dbSNP
rs1003007098	970	dbSNP
rs1035215254	972	dbSNP
rs960482541	973	dbSNP
rs552827612	977	dbSNP
rs1448545066	979	dbSNP
rs1166329926	995	dbSNP
rs1040070004	999	dbSNP
rs923123443	1000	dbSNP
rs1192496010	1007	dbSNP
rs931838168	1013	dbSNP
rs1050204748	1017	dbSNP
rs1209431777	1020	dbSNP
rs1020564468	1021	dbSNP
rs568929940	1025	dbSNP
rs554178580	1032	dbSNP
rs779051041	1040	dbSNP
rs1316137606	1042	dbSNP
rs1216691941	1047	dbSNP
rs1163193967	1058	dbSNP
rs921153440	1062	dbSNP
rs1381884009	1063	dbSNP
rs1269283027	1067	dbSNP
rs1233332950	1069	dbSNP
rs1389156956	1086	dbSNP
rs1356889182	1090	dbSNP
rs192908788	1094	dbSNP
rs1414032322	1111	dbSNP
rs1355045940	1116	dbSNP
rs1330596280	1120	dbSNP
rs1408026556	1131	dbSNP
rs941938205	1138	dbSNP
rs1321357363	1144	dbSNP
rs1394573143	1156	dbSNP
rs1170282041	1163	dbSNP
rs1038374944	1167	dbSNP
rs543134767	1168	dbSNP
rs1364506007	1174	dbSNP
rs35752355	1176	dbSNP
rs912678200	1194	dbSNP
rs940071715	1194	dbSNP
rs986644511	1194	dbSNP
rs561208445	1195	dbSNP
rs994266124	1202	dbSNP
rs1207039984	1206	dbSNP
rs1436473094	1207	dbSNP
rs898513086	1208	dbSNP
rs1024372724	1220	dbSNP
rs1213997977	1224	dbSNP
rs1317410708	1244	dbSNP
rs376086887	1248	dbSNP
rs1323295101	1251	dbSNP
rs1232187452	1265	dbSNP
rs1245158232	1266	dbSNP
rs907269721	1268	dbSNP
rs371163511	1275	dbSNP
rs1222271723	1276	dbSNP
rs931280644	1278	dbSNP
rs1045050073	1279	dbSNP
rs1330914021	1292	dbSNP
rs1462108791	1294	dbSNP
rs750812847	1295	dbSNP
rs1354226726	1297	dbSNP
rs200315859	1305	dbSNP
rs1404861016	1308	dbSNP
rs879472903	1317	dbSNP
rs1464041701	1319	dbSNP
rs906104977	1322	dbSNP
rs1001735012	1323	dbSNP
rs141956388	1327	dbSNP
rs957684766	1328	dbSNP
rs1469504548	1340	dbSNP
rs1035842425	1347	dbSNP
rs896269117	1348	dbSNP
rs1286253137	1351	dbSNP
rs990772216	1351	dbSNP
rs1222057151	1365	dbSNP
rs1019960426	1374	dbSNP
rs1187223841	1385	dbSNP
rs1009284109	1388	dbSNP
rs1455841897	1389	dbSNP
rs1373407128	1390	dbSNP
rs1324186910	1391	dbSNP
rs1160546551	1405	dbSNP
rs1388071838	1413	dbSNP
rs1161394548	1425	dbSNP
rs967422473	1451	dbSNP
rs1204195743	1454	dbSNP
rs1399400131	1456	dbSNP
rs1380263103	1460	dbSNP
rs544050339	1465	dbSNP
rs116651264	1481	dbSNP
rs979088196	1481	dbSNP
rs542204417	1482	dbSNP
rs1380105752	1485	dbSNP
rs1028693802	1486	dbSNP
rs953985566	1490	dbSNP
rs1213041087	1491	dbSNP
rs987061838	1511	dbSNP
rs1280981630	1514	dbSNP
rs16888358	1515	dbSNP
rs908999278	1521	dbSNP
rs939808153	1523	dbSNP
rs972833342	1524	dbSNP
rs1362927299	1527	dbSNP
rs1311795891	1528	dbSNP
rs562549446	1533	dbSNP
rs919970033	1540	dbSNP
rs1314068743	1549	dbSNP
rs1436962304	1555	dbSNP
rs1393717789	1556	dbSNP
rs71813980	1568	dbSNP
rs76264878	1568	dbSNP
rs1428670329	1569	dbSNP
rs1044398329	1570	dbSNP
rs1184205297	1573	dbSNP
rs1477237432	1580	dbSNP
rs906130061	1582	dbSNP
rs938931449	1586	dbSNP
rs1484659377	1594	dbSNP
rs1255131392	1597	dbSNP
rs941966632	1598	dbSNP
rs891981381	1611	dbSNP
rs1298067899	1615	dbSNP
rs1212852333	1618	dbSNP
rs1010320738	1623	dbSNP
rs1294426795	1626	dbSNP
rs1036661554	1643	dbSNP
rs897172994	1645	dbSNP
rs201857930	1657	dbSNP
rs929985063	1673	dbSNP
rs1462833385	1679	dbSNP
rs1355898849	1682	dbSNP
rs1167929538	1683	dbSNP
rs903606094	1692	dbSNP
rs150099066	1719	dbSNP
rs907323399	1721	dbSNP
rs747494130	1724	dbSNP
rs1255419094	1726	dbSNP
rs1481518671	1735	dbSNP
rs560124581	1735	dbSNP
rs1411781289	1737	dbSNP
rs184645942	1741	dbSNP
rs893263073	1742	dbSNP
rs1011847282	1743	dbSNP
rs111245840	1744	dbSNP
rs1235246192	1757	dbSNP
rs972552055	1757	dbSNP
rs1339174666	1767	dbSNP
rs920045180	1771	dbSNP
rs967588528	1772	dbSNP
rs551952010	1775	dbSNP
rs980536950	1780	dbSNP
rs1464720117	1781	dbSNP
rs927329666	1784	dbSNP
rs1166409169	1793	dbSNP
rs1459461428	1793	dbSNP
rs189012194	1801	dbSNP
rs1159831484	1806	dbSNP
rs1029949344	1807	dbSNP
rs1177776008	1813	dbSNP
rs1457449387	1823	dbSNP
rs953134866	1831	dbSNP
rs1253889702	1832	dbSNP
rs1411363775	1836	dbSNP
rs946254122	1837	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.