ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uuccucguuccgccGUAGAGa 5' |||||| Target 5' tcttggtgcttacaCATCTCa 3'	1494 - 1514	120.00	-9.30
2	miRNA 3' uuccucGUUCCGCCGUAGAGa 5' |||||| ||| :|: Target 5' gcacccCAAGGCTGCAGTTTg 3'	139 - 159	103.00	-10.70
3	miRNA 3' uuccUCGUUC---CGCCGUAGAGa 5' :||| | || |:|| || Target 5' tcacGGCACGCTTGC-GTATGTCt 3'	1241 - 1263	100.00	-8.60

Mutant ID	Mutant Position	Mutant Source
COSN8664914	3	COSMIC
COSN8864112	4	COSMIC
COSN8586766	8	COSMIC
COSN30518523	10	COSMIC
COSN30451110	11	COSMIC
COSN30464442	20	COSMIC
COSN30119951	26	COSMIC
COSN17038235	27	COSMIC
COSN13419862	57	COSMIC
COSN30473321	57	COSMIC
COSN17916868	66	COSMIC
COSN17919922	68	COSMIC
COSN30168292	77	COSMIC
COSN30527597	113	COSMIC
COSN30102256	114	COSMIC
COSN30475343	120	COSMIC
COSN30187917	128	COSMIC
COSN30451806	134	COSMIC
COSN30480737	145	COSMIC
COSN28649062	154	COSMIC
COSN31569330	159	COSMIC
COSN21673802	163	COSMIC
COSN31486962	222	COSMIC
COSN28690380	248	COSMIC
COSN28691391	249	COSMIC
COSN9478099	312	COSMIC
COSN19770663	386	COSMIC
COSN5865736	435	COSMIC
COSN30543367	450	COSMIC
COSN26117643	521	COSMIC
COSN17048537	668	COSMIC
COSN5719571	818	COSMIC
COSN22672961	822	COSMIC
COSN8664913	935	COSMIC
COSN9478095	1126	COSMIC
COSN1587017	1343	COSMIC
COSN9478094	1451	COSMIC
COSN22304689	1458	COSMIC
COSN16421145	1725	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs748808768	1	dbSNP
rs754761291	3	dbSNP
rs3741856	4	dbSNP
rs1276628471	5	dbSNP
rs1234064542	6	dbSNP
rs747924029	10	dbSNP
rs1324205785	17	dbSNP
rs1449438191	22	dbSNP
rs780991822	26	dbSNP
rs1229266790	27	dbSNP
rs754867022	27	dbSNP
rs1408665371	29	dbSNP
rs1427887143	39	dbSNP
rs1173814326	40	dbSNP
rs1354378618	47	dbSNP
rs1212128365	50	dbSNP
rs1437607314	63	dbSNP
rs11055941	66	dbSNP
rs11055940	68	dbSNP
rs1483152043	69	dbSNP
rs386760044	69	dbSNP
rs895757890	78	dbSNP
rs1366172951	86	dbSNP
rs1431379495	88	dbSNP
rs1473523803	88	dbSNP
rs34707191	89	dbSNP
rs748909101	89	dbSNP
rs1199938305	90	dbSNP
rs1481203398	95	dbSNP
rs779039098	101	dbSNP
rs1249102912	106	dbSNP
rs764170122	109	dbSNP
rs753866049	113	dbSNP
rs1338762655	118	dbSNP
rs1247167945	121	dbSNP
rs1284859705	125	dbSNP
rs904259746	127	dbSNP
rs1277165153	128	dbSNP
rs1483610769	130	dbSNP
rs1209682015	136	dbSNP
rs1253718847	138	dbSNP
rs776007326	143	dbSNP
rs1252570609	149	dbSNP
rs1208621674	153	dbSNP
rs12301343	154	dbSNP
rs1187563095	158	dbSNP
rs1241037472	164	dbSNP
rs1447536163	166	dbSNP
rs1302376919	167	dbSNP
rs1170453928	169	dbSNP
rs932449809	171	dbSNP
rs183657582	179	dbSNP
rs1433930540	181	dbSNP
rs1172207954	182	dbSNP
rs1400253850	191	dbSNP
rs1398020704	193	dbSNP
rs1334863052	196	dbSNP
rs1225788012	199	dbSNP
rs777038824	216	dbSNP
rs1433491486	217	dbSNP
rs1300514243	222	dbSNP
rs553059866	223	dbSNP
rs1350721586	225	dbSNP
rs1287916042	234	dbSNP
rs1040737811	257	dbSNP
rs542684494	258	dbSNP
rs1200703968	266	dbSNP
rs1282900967	273	dbSNP
rs1301241948	279	dbSNP
rs1464459331	280	dbSNP
rs573775332	283	dbSNP
rs982924774	288	dbSNP
rs1196409907	289	dbSNP
rs1379178768	292	dbSNP
rs1467739191	297	dbSNP
rs1177101075	303	dbSNP
rs746667131	310	dbSNP
rs372594047	311	dbSNP
rs11055938	312	dbSNP
rs1455766578	316	dbSNP
rs4883446	319	dbSNP
rs1366477244	331	dbSNP
rs1386725131	332	dbSNP
rs1180934272	343	dbSNP
rs919974768	351	dbSNP
rs1333228660	352	dbSNP
rs1233488606	357	dbSNP
rs980845241	358	dbSNP
rs1326455202	362	dbSNP
rs969540325	371	dbSNP
rs1025378928	381	dbSNP
rs992203226	383	dbSNP
rs1894820	386	dbSNP
rs1031570511	388	dbSNP
rs998609333	388	dbSNP
rs1264246654	391	dbSNP
rs1206152030	394	dbSNP
rs4883445	399	dbSNP
rs1267905078	400	dbSNP
rs4883444	401	dbSNP
rs386760043	402	dbSNP
rs1337798173	404	dbSNP
rs1894821	407	dbSNP
rs899607915	408	dbSNP
rs1395480949	411	dbSNP
rs1380022136	422	dbSNP
rs1393308558	431	dbSNP
rs1337238398	445	dbSNP
rs1041079841	449	dbSNP
rs770757183	450	dbSNP
rs1348178679	451	dbSNP
rs1229877172	461	dbSNP
rs1288386632	466	dbSNP
rs1158232179	471	dbSNP
rs1320636523	480	dbSNP
rs1242491761	481	dbSNP
rs878862386	489	dbSNP
rs1453967890	493	dbSNP
rs1267212051	497	dbSNP
rs191070404	498	dbSNP
rs931588202	502	dbSNP
rs1482171800	505	dbSNP
rs1184657327	511	dbSNP
rs1412826171	512	dbSNP
rs1423262628	515	dbSNP
rs1163301831	516	dbSNP
rs920089231	522	dbSNP
rs1422684396	529	dbSNP
rs980988126	534	dbSNP
rs1243219553	536	dbSNP
rs948123067	537	dbSNP
rs1308206103	539	dbSNP
rs1894822	539	dbSNP
rs1226049520	540	dbSNP
rs1314238958	552	dbSNP
rs992548030	563	dbSNP
rs1463978473	568	dbSNP
rs1272253526	574	dbSNP
rs1343982949	578	dbSNP
rs1247765597	586	dbSNP
rs185281337	590	dbSNP
rs956737574	592	dbSNP
rs1180251105	593	dbSNP
rs1258323927	596	dbSNP
rs1424941772	600	dbSNP
rs923976249	625	dbSNP
rs534614595	626	dbSNP
rs1431949124	630	dbSNP
rs1168840194	631	dbSNP
rs968627578	633	dbSNP
rs1445334070	650	dbSNP
rs1304123657	655	dbSNP
rs1379217658	657	dbSNP
rs1434313429	659	dbSNP
rs1295241688	660	dbSNP
rs1367103317	671	dbSNP
rs1228958278	682	dbSNP
rs1021399028	685	dbSNP
rs1311881516	686	dbSNP
rs144056951	687	dbSNP
rs1360183938	691	dbSNP
rs996704693	698	dbSNP
rs1188499956	701	dbSNP
rs1246653181	704	dbSNP
rs182841411	713	dbSNP
rs1317323539	720	dbSNP
rs1894824	723	dbSNP
rs144744879	731	dbSNP
rs1424868394	732	dbSNP
rs1319617559	736	dbSNP
rs1001909800	741	dbSNP
rs886766812	750	dbSNP
rs1367341246	752	dbSNP
rs766782221	758	dbSNP
rs1457104350	761	dbSNP
rs1365175593	769	dbSNP
rs1217711845	773	dbSNP
rs995741350	774	dbSNP
rs1165003055	775	dbSNP
rs751927083	776	dbSNP
rs1208259913	786	dbSNP
rs898779080	787	dbSNP
rs1390049926	792	dbSNP
rs1194562994	794	dbSNP
rs1449034299	798	dbSNP
rs1265725434	799	dbSNP
rs1209453452	800	dbSNP
rs1490594496	803	dbSNP
rs549119305	814	dbSNP
rs1429641845	817	dbSNP
rs1045614625	822	dbSNP
rs1156383062	827	dbSNP
rs773033729	831	dbSNP
rs1369487786	832	dbSNP
rs762664038	852	dbSNP
rs1226335433	854	dbSNP
rs1322757517	858	dbSNP
rs1341570859	865	dbSNP
rs748134219	867	dbSNP
rs1269482353	869	dbSNP
rs1292973007	870	dbSNP
rs747709281	871	dbSNP
rs917958363	879	dbSNP
rs1056563111	880	dbSNP
rs1224843434	889	dbSNP
rs867178250	902	dbSNP
rs1321406443	906	dbSNP
rs1224317663	918	dbSNP
rs1270023764	922	dbSNP
rs761765219	923	dbSNP
rs1413559459	924	dbSNP
rs1470147016	928	dbSNP
rs1162592113	932	dbSNP
rs1411619794	945	dbSNP
rs935454649	947	dbSNP
rs1164027638	959	dbSNP
rs1369678813	961	dbSNP
rs1365864126	963	dbSNP
rs1409473594	964	dbSNP
rs1304610293	967	dbSNP
rs923923953	970	dbSNP
rs979429079	973	dbSNP
rs190162193	975	dbSNP
rs1427221923	989	dbSNP
rs968576472	991	dbSNP
rs1369410889	992	dbSNP
rs535570353	1003	dbSNP
rs975611584	1009	dbSNP
rs749260089	1011	dbSNP
rs1376279710	1016	dbSNP
rs770722149	1021	dbSNP
rs963919630	1023	dbSNP
rs1186297349	1036	dbSNP
rs1198649957	1066	dbSNP
rs1480872349	1075	dbSNP
rs1473757532	1093	dbSNP
rs1164084652	1101	dbSNP
rs1019899354	1107	dbSNP
rs11055935	1110	dbSNP
rs1203315743	1111	dbSNP
rs1398985892	1130	dbSNP
rs1410726331	1132	dbSNP
rs951134847	1139	dbSNP
rs1338735611	1140	dbSNP
rs1455076436	1142	dbSNP
rs1285927443	1149	dbSNP
rs1316274927	1153	dbSNP
rs7132811	1155	dbSNP
rs1273820478	1184	dbSNP
rs1339716874	1192	dbSNP
rs1199494408	1196	dbSNP
rs1275928093	1202	dbSNP
rs768911947	1202	dbSNP
rs1443589380	1207	dbSNP
rs995185761	1208	dbSNP
rs185568570	1212	dbSNP
rs780061081	1220	dbSNP
rs898726839	1240	dbSNP
rs145505477	1244	dbSNP
rs754956753	1245	dbSNP
rs1345947270	1246	dbSNP
rs781735075	1249	dbSNP
rs896647467	1250	dbSNP
rs796922630	1251	dbSNP
rs1353405831	1255	dbSNP
rs1329028086	1256	dbSNP
rs1057056365	1258	dbSNP
rs1470958469	1265	dbSNP
rs1412104002	1272	dbSNP
rs1388621028	1278	dbSNP
rs1400750886	1289	dbSNP
rs766955572	1290	dbSNP
rs752014628	1291	dbSNP
rs1043682691	1300	dbSNP
rs1180214174	1306	dbSNP
rs946721011	1312	dbSNP
rs1472419990	1320	dbSNP
rs1233519012	1322	dbSNP
rs914439742	1323	dbSNP
rs975349106	1333	dbSNP
rs1440795128	1345	dbSNP
rs1276371390	1359	dbSNP
rs942652083	1367	dbSNP
rs1376205792	1370	dbSNP
rs774114718	1371	dbSNP
rs912379793	1393	dbSNP
rs986661493	1395	dbSNP
rs951083081	1397	dbSNP
rs1202966444	1401	dbSNP
rs1291089874	1403	dbSNP
rs78017164	1406	dbSNP
rs765856567	1413	dbSNP
rs139357414	1418	dbSNP
rs1347063277	1425	dbSNP
rs1223715769	1427	dbSNP
rs1226188960	1447	dbSNP
rs962380040	1448	dbSNP
rs1241116099	1451	dbSNP
rs1372211941	1455	dbSNP
rs1279613724	1457	dbSNP
rs1023758765	1464	dbSNP
rs1012480198	1467	dbSNP
rs1336568059	1476	dbSNP
rs1251200230	1478	dbSNP
rs868669122	1484	dbSNP
rs1161376290	1494	dbSNP
rs1453994609	1495	dbSNP
rs1035163666	1501	dbSNP
rs1161872772	1502	dbSNP
rs999665618	1515	dbSNP
rs902566443	1516	dbSNP
rs1157488700	1518	dbSNP
rs1348092435	1520	dbSNP
rs1392694196	1536	dbSNP
rs1307069180	1544	dbSNP
rs1043796964	1553	dbSNP
rs1293708025	1556	dbSNP
rs946835597	1558	dbSNP
rs1223513044	1560	dbSNP
rs55754582	1563	dbSNP
rs1471813720	1574	dbSNP
rs1479774734	1579	dbSNP
rs1179076579	1588	dbSNP
rs182067296	1596	dbSNP
rs762514997	1601	dbSNP
rs144721737	1602	dbSNP
rs1423780171	1603	dbSNP
rs942620644	1606	dbSNP
rs912483502	1607	dbSNP
rs1425620427	1610	dbSNP
rs1168349580	1613	dbSNP
rs1244718050	1615	dbSNP
rs759106780	1616	dbSNP
rs753247539	1649	dbSNP
rs35798487	1652	dbSNP
rs986608817	1654	dbSNP
rs1206645195	1655	dbSNP
rs775537666	1664	dbSNP
rs1316142159	1666	dbSNP
rs1383261425	1669	dbSNP
rs1227475068	1674	dbSNP
rs147546883	1687	dbSNP
rs1206847096	1688	dbSNP
rs1225114078	1689	dbSNP
rs1353570356	1696	dbSNP
rs1313813232	1704	dbSNP
rs918308421	1706	dbSNP
rs1445749957	1717	dbSNP
rs1188381729	1723	dbSNP
rs1385670886	1724	dbSNP
rs1318927557	1726	dbSNP
rs973813348	1729	dbSNP
rs1388675085	1732	dbSNP
rs1429961647	1735	dbSNP
rs962797608	1735	dbSNP
rs1358080870	1744	dbSNP
rs144886062	1758	dbSNP
rs1338123020	1759	dbSNP
rs1346165171	1760	dbSNP
rs990903189	1765	dbSNP
rs1403871120	1778	dbSNP
rs1384686093	1789	dbSNP
rs960962913	1790	dbSNP
rs1363759268	1798	dbSNP
rs1216564010	1799	dbSNP
rs771847561	1802	dbSNP
rs1038158011	1803	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.