ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ggguGGUCGACGAGGGACCGGGu 5' :|| || | ||||||||| Target 5' tctcTCACCT-C-CCCTGGCCCt 3'	568 - 588	157.00	-27.20
2	miRNA 3' ggguGGUCGAC-GAGGGACCGGGu 5' ||: | | || :||||:|| Target 5' gagtCCGACAGACTGTCTGGTCCc 3'	1092 - 1115	139.00	-24.60
3	miRNA 3' ggguGGUCGA-CGAGGGACCGGGu 5' |: ||| ||||| ||||| Target 5' atgcCTTGCTCACTCCCAGGCCCg 3'	162 - 185	135.00	-25.20

Mutant ID	Mutant Position	Mutant Source
COSN30500069	3	COSMIC
COSN26993730	10	COSMIC
COSN28847781	30	COSMIC
COSN30478894	33	COSMIC
COSN30642955	40	COSMIC
COSN28749651	42	COSMIC
COSN26993729	43	COSMIC
COSN31532844	64	COSMIC
COSN26993731	75	COSMIC
COSN31498356	77	COSMIC
COSN30457585	83	COSMIC
COSN31486328	85	COSMIC
COSN30756192	143	COSMIC
COSN30126737	159	COSMIC
COSN32070642	188	COSMIC
COSN32070643	199	COSMIC
COSN28877437	243	COSMIC
COSN5802194	425	COSMIC
COSN31540156	579	COSMIC
COSN31606657	619	COSMIC
COSN1215481	854	COSMIC
COSN32056009	1181	COSMIC
COSN7126722	1183	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs372726169	2	dbSNP
rs751065965	3	dbSNP
rs770545995	4	dbSNP
rs1369569357	6	dbSNP
rs1359791731	11	dbSNP
rs376021198	18	dbSNP
rs571961567	19	dbSNP
rs752206859	20	dbSNP
rs762425347	23	dbSNP
rs767425225	26	dbSNP
rs750887873	32	dbSNP
rs1162507256	36	dbSNP
rs750581276	37	dbSNP
rs113782061	40	dbSNP
rs766562882	41	dbSNP
rs370612551	43	dbSNP
rs555706421	44	dbSNP
rs1180146361	47	dbSNP
rs371533253	48	dbSNP
rs1320344150	49	dbSNP
rs1252827328	50	dbSNP
rs758394266	53	dbSNP
rs1457773481	61	dbSNP
rs1320111513	62	dbSNP
rs893080045	63	dbSNP
rs757609205	64	dbSNP
rs942855390	65	dbSNP
rs1229200198	70	dbSNP
rs1010540546	74	dbSNP
rs1020187996	75	dbSNP
rs974198909	76	dbSNP
rs547795715	82	dbSNP
rs971827942	83	dbSNP
rs1308102610	89	dbSNP
rs1443972972	90	dbSNP
rs750311470	97	dbSNP
rs919714003	105	dbSNP
rs1322672925	111	dbSNP
rs1405698153	114	dbSNP
rs1415879102	117	dbSNP
rs1034881219	125	dbSNP
rs1472215789	126	dbSNP
rs1447936856	133	dbSNP
rs573930366	134	dbSNP
rs746827981	135	dbSNP
rs770726763	136	dbSNP
rs761233629	137	dbSNP
rs776249741	142	dbSNP
rs1222781483	144	dbSNP
rs1352504884	146	dbSNP
rs745557579	147	dbSNP
rs769514346	148	dbSNP
rs775145675	154	dbSNP
rs1434556119	155	dbSNP
rs1186763304	156	dbSNP
rs1243625976	158	dbSNP
rs1446252068	159	dbSNP
rs762550050	161	dbSNP
rs1379970779	166	dbSNP
rs930239128	170	dbSNP
rs1160435667	177	dbSNP
rs1224917131	181	dbSNP
rs544631775	182	dbSNP
rs1413931788	184	dbSNP
rs562963578	185	dbSNP
rs755849651	186	dbSNP
rs944406673	188	dbSNP
rs533512209	189	dbSNP
rs900257381	193	dbSNP
rs1334875447	208	dbSNP
rs754088929	209	dbSNP
rs1280864515	210	dbSNP
rs1342286158	219	dbSNP
rs1199346463	220	dbSNP
rs905082074	221	dbSNP
rs1000655322	222	dbSNP
rs1054303217	223	dbSNP
rs1196586298	225	dbSNP
rs1237336857	229	dbSNP
rs755229356	231	dbSNP
rs1184278350	232	dbSNP
rs1405790219	232	dbSNP
rs545572591	236	dbSNP
rs1376092760	237	dbSNP
rs1032132492	238	dbSNP
rs560678482	239	dbSNP
rs1409021595	241	dbSNP
rs766106869	242	dbSNP
rs1331190868	245	dbSNP
rs1397677266	247	dbSNP
rs1277279484	248	dbSNP
rs1393447962	251	dbSNP
rs1330506004	254	dbSNP
rs1010054130	258	dbSNP
rs1042067335	262	dbSNP
rs753403273	264	dbSNP
rs1365846585	268	dbSNP
rs1290503219	274	dbSNP
rs1003293221	282	dbSNP
rs1358727573	288	dbSNP
rs1005007540	304	dbSNP
rs1161713225	308	dbSNP
rs1436867107	310	dbSNP
rs955415821	312	dbSNP
rs1409811251	313	dbSNP
rs527953350	320	dbSNP
rs760702766	321	dbSNP
rs964287083	323	dbSNP
rs974274170	324	dbSNP
rs1360843637	328	dbSNP
rs1014995717	331	dbSNP
rs1483206165	332	dbSNP
rs1254954328	335	dbSNP
rs920081002	337	dbSNP
rs1445460242	338	dbSNP
rs1302082278	342	dbSNP
rs1373843971	344	dbSNP
rs1034273537	354	dbSNP
rs958800329	358	dbSNP
rs1394263934	360	dbSNP
rs1349318293	361	dbSNP
rs1378315420	362	dbSNP
rs929740774	375	dbSNP
rs549432690	377	dbSNP
rs1416654253	385	dbSNP
rs1368950272	387	dbSNP
rs192191869	392	dbSNP
rs566173270	408	dbSNP
rs917627814	412	dbSNP
rs77539404	422	dbSNP
rs1208402484	423	dbSNP
rs147919375	424	dbSNP
rs912816280	425	dbSNP
rs1188055174	434	dbSNP
rs1486334796	447	dbSNP
rs1248973555	449	dbSNP
rs753220231	453	dbSNP
rs549405797	454	dbSNP
rs183135492	457	dbSNP
rs1472274630	461	dbSNP
rs1252908180	471	dbSNP
rs1191854835	472	dbSNP
rs1053579760	477	dbSNP
rs1321075574	478	dbSNP
rs976173931	480	dbSNP
rs892280092	487	dbSNP
rs921571641	488	dbSNP
rs1348768561	494	dbSNP
rs931624154	495	dbSNP
rs1412662899	500	dbSNP
rs1410711503	502	dbSNP
rs1398848423	503	dbSNP
rs1009790457	508	dbSNP
rs1175320392	514	dbSNP
rs758967208	517	dbSNP
rs1369564374	519	dbSNP
rs1054230945	528	dbSNP
rs778235797	531	dbSNP
rs891166482	552	dbSNP
rs945905723	565	dbSNP
rs1041517603	570	dbSNP
rs1252174708	576	dbSNP
rs867850219	580	dbSNP
rs1436521261	584	dbSNP
rs1488000593	585	dbSNP
rs1273564457	597	dbSNP
rs1215555489	599	dbSNP
rs1276127322	608	dbSNP
rs1364940639	611	dbSNP
rs907116660	611	dbSNP
rs1002769535	613	dbSNP
rs1055732701	619	dbSNP
rs1391991116	628	dbSNP
rs1328310630	635	dbSNP
rs1456163343	640	dbSNP
rs1388491453	649	dbSNP
rs894506910	651	dbSNP
rs1470137142	656	dbSNP
rs995446525	657	dbSNP
rs1008271110	658	dbSNP
rs951464192	659	dbSNP
rs1207763380	663	dbSNP
rs1272673409	668	dbSNP
rs1195242297	670	dbSNP
rs1018280838	672	dbSNP
rs747446880	678	dbSNP
rs964319977	683	dbSNP
rs1353009718	684	dbSNP
rs79187519	690	dbSNP
rs76996310	691	dbSNP
rs995742915	703	dbSNP
rs1232863793	712	dbSNP
rs1331881719	721	dbSNP
rs538347174	724	dbSNP
rs960728150	729	dbSNP
rs1019759265	736	dbSNP
rs1433292150	739	dbSNP
rs1322214779	747	dbSNP
rs966029875	749	dbSNP
rs1027628052	752	dbSNP
rs1248494449	763	dbSNP
rs1477118273	765	dbSNP
rs187814571	768	dbSNP
rs951592725	769	dbSNP
rs975691246	770	dbSNP
rs921497665	772	dbSNP
rs796431803	776	dbSNP
rs36270	779	dbSNP
rs917674689	786	dbSNP
rs1451026411	787	dbSNP
rs1448632462	791	dbSNP
rs970879564	799	dbSNP
rs1166515781	804	dbSNP
rs980438180	813	dbSNP
rs945776359	816	dbSNP
rs1041959222	817	dbSNP
rs938497784	822	dbSNP
rs1271130180	826	dbSNP
rs558744482	828	dbSNP
rs754029238	835	dbSNP
rs1325164032	838	dbSNP
rs536477280	841	dbSNP
rs1347608173	844	dbSNP
rs1407563074	847	dbSNP
rs1053648131	850	dbSNP
rs913774530	852	dbSNP
rs1323997559	854	dbSNP
rs1435201912	858	dbSNP
rs945232785	861	dbSNP
rs1175190084	871	dbSNP
rs1427420677	874	dbSNP
rs554553583	881	dbSNP
rs1168262894	882	dbSNP
rs891244762	895	dbSNP
rs576329003	896	dbSNP
rs1295894683	897	dbSNP
rs1339180235	906	dbSNP
rs1168512465	907	dbSNP
rs370477282	913	dbSNP
rs1292355396	914	dbSNP
rs1274207411	915	dbSNP
rs1216641837	917	dbSNP
rs1449874645	922	dbSNP
rs1284063260	952	dbSNP
rs1020106973	955	dbSNP
rs901364997	964	dbSNP
rs1314149057	966	dbSNP
rs997014639	972	dbSNP
rs1231040585	974	dbSNP
rs1353515257	978	dbSNP
rs1039753558	989	dbSNP
rs1411386253	995	dbSNP
rs1248525443	1013	dbSNP
rs141754139	1020	dbSNP
rs1162647001	1029	dbSNP
rs770006585	1046	dbSNP
rs1475490539	1049	dbSNP
rs1420161467	1057	dbSNP
rs1389288758	1061	dbSNP
rs1472399027	1063	dbSNP
rs1473043055	1071	dbSNP
rs1027243978	1080	dbSNP
rs1233626946	1081	dbSNP
rs1207080927	1090	dbSNP
rs1450303002	1092	dbSNP
rs190334079	1099	dbSNP
rs1337560559	1104	dbSNP
rs763944908	1106	dbSNP
rs1226001095	1110	dbSNP
rs545535649	1112	dbSNP
rs1347968603	1114	dbSNP
rs1443903165	1114	dbSNP
rs967545243	1121	dbSNP
rs1461943088	1124	dbSNP
rs1396537215	1137	dbSNP
rs1159303496	1153	dbSNP
rs1004421555	1154	dbSNP
rs977224301	1156	dbSNP
rs1025554080	1160	dbSNP
rs1456712493	1161	dbSNP
rs970480007	1163	dbSNP
rs560640005	1166	dbSNP
rs1477144404	1169	dbSNP
rs1246538730	1170	dbSNP
rs926349953	1180	dbSNP
rs572523932	1181	dbSNP
rs182893669	1193	dbSNP
rs375917637	1207	dbSNP
rs1315877311	1208	dbSNP
rs1284345362	1215	dbSNP
rs915832491	1217	dbSNP
rs913859714	1218	dbSNP
rs1354546045	1219	dbSNP
rs945278995	1220	dbSNP
rs1268827465	1225	dbSNP
rs1429933838	1227	dbSNP
rs1048397430	1229	dbSNP
rs775644806	1237	dbSNP
rs911697207	1241	dbSNP
rs1394659826	1250	dbSNP
rs763111585	1260	dbSNP
rs1174657281	1264	dbSNP
rs1442730699	1265	dbSNP
rs1189372481	1270	dbSNP
rs1486463598	1271	dbSNP
rs939998479	1271	dbSNP
rs561413612	1272	dbSNP
rs1217251745	1277	dbSNP
rs899886033	1278	dbSNP
rs375170574	1280	dbSNP
rs901245980	1295	dbSNP
rs1413522771	1296	dbSNP
rs1455239369	1306	dbSNP
rs997026085	1309	dbSNP
rs1157507621	1310	dbSNP
rs1384874505	1318	dbSNP
rs772202618	1321	dbSNP
rs751509287	1323	dbSNP
rs916546600	1331	dbSNP
rs1290739507	1338	dbSNP
rs532076426	1354	dbSNP
rs1445826160	1355	dbSNP
rs1375081073	1358	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.