ID	Duplex structure	Position	Score	MFE
1	miRNA 3' acauCACGUUGAUACGUUUUGa 5' ||| |||| |||||| Target 5' atctGTGAAACTC--CAAAACa 3'	770 - 789	133.00	-7.60
2	miRNA 3' acaucacguuGAUACGUUUUGa 5' | | |||||:| Target 5' gcctcccctcCCAAGCAAAGCt 3'	233 - 254	128.00	-7.40
3	miRNA 3' acaUCACG-UUGA-UACGUUUUGa 5' ||| : ::|| |||:||:|| Target 5' caaAGTTTGGGCTGATGTAAGACt 3'	354 - 377	127.00	-10.90

Mutant ID	Mutant Position	Mutant Source
COSM4150319	17	COSMIC
COSM7720509	34	COSMIC
COSM8847814	46	COSMIC
COSM7667186	78	COSMIC
COSM8199915	93	COSMIC
COSM8104895	120	COSMIC
COSM7299096	128	COSMIC
COSM7692868	142	COSMIC
COSM9534429	144	COSMIC
COSM8531097	152	COSMIC
COSM6334926	164	COSMIC
COSN31556068	211	COSMIC
COSN22394474	374	COSMIC
COSN24304467	974	COSMIC
COSN31607123	974	COSMIC
COSN26584326	1000	COSMIC
COSN19348249	1064	COSMIC
COSN26575296	1167	COSMIC
COSN26506368	1168	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1390032409	3	dbSNP
rs1306725404	4	dbSNP
rs754239625	4	dbSNP
rs367576053	6	dbSNP
rs756751181	7	dbSNP
rs1189251120	9	dbSNP
rs1485696962	11	dbSNP
rs1000314823	16	dbSNP
rs1214337871	18	dbSNP
rs1256148820	19	dbSNP
rs753222876	23	dbSNP
rs766034385	27	dbSNP
rs1284948994	30	dbSNP
rs762548623	31	dbSNP
rs142697690	34	dbSNP
rs769206581	37	dbSNP
rs1017642173	38	dbSNP
rs1176464545	41	dbSNP
rs764948095	42	dbSNP
rs761314786	44	dbSNP
rs1008793031	47	dbSNP
rs375317183	50	dbSNP
rs768439179	52	dbSNP
rs1047523660	54	dbSNP
rs1346304030	55	dbSNP
rs1461518720	56	dbSNP
rs747480158	58	dbSNP
rs775385985	61	dbSNP
rs899045690	65	dbSNP
rs989714156	75	dbSNP
rs771954100	84	dbSNP
rs568681316	85	dbSNP
rs1298009329	86	dbSNP
rs1416737821	92	dbSNP
rs771237731	94	dbSNP
rs749466052	97	dbSNP
rs1397700788	98	dbSNP
rs778321232	99	dbSNP
rs756524219	105	dbSNP
rs937783477	106	dbSNP
rs753061109	109	dbSNP
rs555244830	110	dbSNP
rs781772898	113	dbSNP
rs766834485	119	dbSNP
rs749964493	122	dbSNP
rs1362812711	124	dbSNP
rs1270097695	126	dbSNP
rs148231637	128	dbSNP
rs761598315	129	dbSNP
rs1309189800	130	dbSNP
rs753461877	131	dbSNP
rs1252068550	137	dbSNP
rs1046499196	139	dbSNP
rs1393257806	140	dbSNP
rs763929999	141	dbSNP
rs1429739204	149	dbSNP
rs780514108	150	dbSNP
rs760488131	159	dbSNP
rs191149538	161	dbSNP
rs1256719907	163	dbSNP
rs775197260	164	dbSNP
rs1804951	172	dbSNP
rs772057203	173	dbSNP
rs759375554	181	dbSNP
rs1026449102	182	dbSNP
rs774553734	191	dbSNP
rs1200803566	201	dbSNP
rs1308336410	210	dbSNP
rs1272320006	216	dbSNP
rs1212898053	230	dbSNP
rs1391572215	234	dbSNP
rs950295136	235	dbSNP
rs1016163006	248	dbSNP
rs1311438799	249	dbSNP
rs1012777374	263	dbSNP
rs376690232	265	dbSNP
rs917491654	265	dbSNP
rs552271215	270	dbSNP
rs934806912	271	dbSNP
rs1296572036	272	dbSNP
rs532551458	276	dbSNP
rs569787218	277	dbSNP
rs773429197	283	dbSNP
rs1279317879	285	dbSNP
rs1804952	286	dbSNP
rs1485983412	288	dbSNP
rs1212196682	292	dbSNP
rs978822314	294	dbSNP
rs1485267385	306	dbSNP
rs1186525632	311	dbSNP
rs1391705604	316	dbSNP
rs549865914	321	dbSNP
rs1452174302	322	dbSNP
rs1172216990	323	dbSNP
rs530578174	325	dbSNP
rs964809382	328	dbSNP
rs1343885004	330	dbSNP
rs893800390	334	dbSNP
rs1379911321	335	dbSNP
rs1321290995	336	dbSNP
rs1367689102	340	dbSNP
rs1157290726	345	dbSNP
rs1302907214	345	dbSNP
rs1017576516	356	dbSNP
rs1220686596	372	dbSNP
rs1282071155	373	dbSNP
rs1316265970	376	dbSNP
rs1215960959	377	dbSNP
rs987885953	389	dbSNP
rs1192650658	396	dbSNP
rs954612669	399	dbSNP
rs1412969520	406	dbSNP
rs1478568540	412	dbSNP
rs943633177	419	dbSNP
rs1406506546	421	dbSNP
rs1026649974	425	dbSNP
rs910758337	431	dbSNP
rs561534897	441	dbSNP
rs1325499207	446	dbSNP
rs541651936	447	dbSNP
rs993464469	473	dbSNP
rs775293466	478	dbSNP
rs1277544395	480	dbSNP
rs898993464	486	dbSNP
rs987665201	487	dbSNP
rs1228896907	488	dbSNP
rs933455969	489	dbSNP
rs1332059225	503	dbSNP
rs771693677	507	dbSNP
rs1212459152	508	dbSNP
rs185988242	512	dbSNP
rs1201534295	518	dbSNP
rs77651612	529	dbSNP
rs1469914119	539	dbSNP
rs1183568751	547	dbSNP
rs1414762966	554	dbSNP
rs963048508	560	dbSNP
rs181655460	565	dbSNP
rs576779013	571	dbSNP
rs1407274416	572	dbSNP
rs1326655084	573	dbSNP
rs1484158061	575	dbSNP
rs949068622	578	dbSNP
rs1258907345	580	dbSNP
rs1290151850	584	dbSNP
rs1207862224	590	dbSNP
rs1353413901	590	dbSNP
rs1232217779	594	dbSNP
rs1284258587	595	dbSNP
rs892164143	601	dbSNP
rs991075483	605	dbSNP
rs1289796125	607	dbSNP
rs769455609	608	dbSNP
rs958654882	612	dbSNP
rs1053766188	616	dbSNP
rs1437631001	617	dbSNP
rs1206236293	625	dbSNP
rs188525242	626	dbSNP
rs1339529350	629	dbSNP
rs1312758345	637	dbSNP
rs7800	638	dbSNP
rs1484970154	641	dbSNP
rs543536393	651	dbSNP
rs1397272332	655	dbSNP
rs1187374436	662	dbSNP
rs778308400	662	dbSNP
rs926086652	664	dbSNP
rs1469764035	668	dbSNP
rs978938777	672	dbSNP
rs943401394	673	dbSNP
rs1419000490	679	dbSNP
rs910630966	680	dbSNP
rs1309612264	686	dbSNP
rs1373617674	695	dbSNP
rs1161854428	697	dbSNP
rs1053791894	698	dbSNP
rs943330793	700	dbSNP
rs987614335	708	dbSNP
rs954868320	712	dbSNP
rs574630198	714	dbSNP
rs1224540841	717	dbSNP
rs1269235071	721	dbSNP
rs1368124481	725	dbSNP
rs1229686588	728	dbSNP
rs1277190273	734	dbSNP
rs770249642	735	dbSNP
rs1006757066	736	dbSNP
rs971929919	740	dbSNP
rs149047015	741	dbSNP
rs889388315	742	dbSNP
rs1444209568	743	dbSNP
rs1191027410	745	dbSNP
rs1241808169	746	dbSNP
rs1450309810	755	dbSNP
rs867965275	757	dbSNP
rs1170307163	760	dbSNP
rs1052423864	761	dbSNP
rs1016015622	765	dbSNP
rs748707496	769	dbSNP
rs535252600	771	dbSNP
rs745564699	771	dbSNP
rs919417259	773	dbSNP
rs971826879	777	dbSNP
rs566204903	789	dbSNP
rs8096	790	dbSNP
rs376756738	791	dbSNP
rs991181984	791	dbSNP
rs958590156	794	dbSNP
rs1035635066	798	dbSNP
rs186575528	799	dbSNP
rs967272723	800	dbSNP
rs1013365102	806	dbSNP
rs1190785291	808	dbSNP
rs1011376082	810	dbSNP
rs892107555	815	dbSNP
rs1052119914	816	dbSNP
rs1052950	820	dbSNP
rs999084688	824	dbSNP
rs74057057	825	dbSNP
rs1388775939	828	dbSNP
rs1458315517	829	dbSNP
rs1323492466	832	dbSNP
rs755344382	840	dbSNP
rs1296017896	841	dbSNP
rs1386626566	845	dbSNP
rs1302393323	847	dbSNP
rs943507923	851	dbSNP
rs910759085	867	dbSNP
rs1052261343	868	dbSNP
rs71445122	872	dbSNP
rs1329955143	873	dbSNP
rs1210598730	880	dbSNP
rs1271579549	883	dbSNP
rs1460393254	891	dbSNP
rs933446465	892	dbSNP
rs919230405	893	dbSNP
rs1269170480	897	dbSNP
rs1417045618	899	dbSNP
rs1412544409	901	dbSNP
rs761924154	901	dbSNP
rs963232705	902	dbSNP
rs569723863	903	dbSNP
rs1393343931	906	dbSNP
rs1390276553	907	dbSNP
rs1458075397	908	dbSNP
rs1036494116	912	dbSNP
rs6786	913	dbSNP
rs1288277529	914	dbSNP
rs181465986	914	dbSNP
rs1230374313	915	dbSNP
rs969609752	916	dbSNP
rs1024838919	918	dbSNP
rs1449960217	920	dbSNP
rs189431465	923	dbSNP
rs928283165	924	dbSNP
rs1204093180	926	dbSNP
rs1252529853	934	dbSNP
rs1483985774	937	dbSNP
rs956500195	941	dbSNP
rs548046935	943	dbSNP
rs1450877115	955	dbSNP
rs1412406362	960	dbSNP
rs1267577397	963	dbSNP
rs1166499694	972	dbSNP
rs1204761351	973	dbSNP
rs1370924662	973	dbSNP
rs1000540475	977	dbSNP
rs1307795979	982	dbSNP
rs903554247	987	dbSNP
rs1412190701	988	dbSNP
rs1378565945	1000	dbSNP
rs967291222	1000	dbSNP
rs1396623434	1004	dbSNP
rs1043324408	1014	dbSNP
rs1315112840	1016	dbSNP
rs1305977186	1017	dbSNP
rs913119600	1017	dbSNP
rs989998434	1018	dbSNP
rs1344278299	1019	dbSNP
rs1208677008	1020	dbSNP
rs1273232433	1020	dbSNP
rs528310799	1021	dbSNP
rs879161351	1027	dbSNP
rs889268226	1030	dbSNP
rs758742005	1033	dbSNP
rs1448481038	1035	dbSNP
rs1444488144	1039	dbSNP
rs1300523120	1044	dbSNP
rs903164894	1055	dbSNP
rs1174594764	1059	dbSNP
rs1431968627	1059	dbSNP
rs1041741699	1061	dbSNP
rs947421306	1063	dbSNP
rs1388687411	1064	dbSNP
rs559340630	1065	dbSNP
rs1360189292	1071	dbSNP
rs957170855	1079	dbSNP
rs1330532139	1080	dbSNP
rs1367122641	1080	dbSNP
rs748392729	1080	dbSNP
rs1312688940	1081	dbSNP
rs61919664	1082	dbSNP
rs1430498613	1083	dbSNP
rs1391931350	1084	dbSNP
rs1464021314	1085	dbSNP
rs1188833701	1086	dbSNP
rs1249442555	1088	dbSNP
rs1451728161	1091	dbSNP
rs1189833750	1092	dbSNP
rs1170791219	1096	dbSNP
rs1477562664	1098	dbSNP
rs1377938491	1100	dbSNP
rs1200774293	1101	dbSNP
rs1435651764	1105	dbSNP
rs1250638682	1106	dbSNP
rs1178368716	1108	dbSNP
rs1205240931	1110	dbSNP
rs1483790442	1111	dbSNP
rs1456729684	1112	dbSNP
rs184448526	1112	dbSNP
rs532346346	1113	dbSNP
rs1391020730	1117	dbSNP
rs1008046236	1121	dbSNP
rs1241653569	1122	dbSNP
rs953443553	1122	dbSNP
rs1030748685	1129	dbSNP
rs1304726502	1131	dbSNP
rs1327884638	1134	dbSNP
rs1208737832	1139	dbSNP
rs1269705368	1140	dbSNP
rs997489879	1141	dbSNP
rs1219885452	1142	dbSNP
rs1246454015	1144	dbSNP
rs1277354566	1146	dbSNP
rs1193448461	1147	dbSNP
rs1218029425	1148	dbSNP
rs898093551	1149	dbSNP
rs1159994440	1153	dbSNP
rs1051958349	1156	dbSNP
rs1403786370	1167	dbSNP
rs933364017	1168	dbSNP
rs1323096213	1170	dbSNP
rs573494596	1171	dbSNP
rs1438281484	1172	dbSNP
rs758501954	1172	dbSNP
rs1349795742	1175	dbSNP
rs1006367997	1176	dbSNP
rs1228529208	1176	dbSNP
rs1353863483	1176	dbSNP
rs149252440	1176	dbSNP
rs545282816	1176	dbSNP
rs1036440140	1180	dbSNP
rs768999546	1184	dbSNP
rs887932565	1190	dbSNP
rs1055581556	1195	dbSNP
rs1483003132	1201	dbSNP
rs1348796019	1213	dbSNP
rs909241812	1226	dbSNP
rs1414320850	1227	dbSNP
rs750510257	1228	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.