ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ggACUCGGAGAGACGGAGACGa 5' | ||| || || ||||| Target 5' aaTTAGC--ATCGGCATCTGCt 3'	30 - 49	124.00	-12.60
2	miRNA 3' ggACUCGGAG----AGACGGAGACGa 5' || |:| | |:| |:||||: Target 5' tgTGTGTCACCACATTTTCTTCTGTg 3'	1035 - 1060	122.00	-9.70
3	miRNA 3' ggACUCGGAGAGAC----GGAGACga 5' ||| ||| || | |:|||| Target 5' agTGA-CCT-TCAGAAGACTTCTGaa 3'	465 - 488	116.00	-11.70

Mutant ID	Mutant Position	Mutant Source
COSN27000406	20	COSMIC
COSN30457264	29	COSMIC
COSN27000401	39	COSMIC
COSN20048246	40	COSMIC
COSN30167285	57	COSMIC
COSN30720197	60	COSMIC
COSN30168121	80	COSMIC
COSN28835641	100	COSMIC
COSN20065272	101	COSMIC
COSN30166198	112	COSMIC
COSN22501716	254	COSMIC
COSN9703680	262	COSMIC
COSN24714383	513	COSMIC
COSN15090362	603	COSMIC
COSN24316704	764	COSMIC
COSN16228500	1106	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs779196250	7	dbSNP
rs552060922	10	dbSNP
rs753928128	25	dbSNP
rs369412614	31	dbSNP
rs756642508	32	dbSNP
rs750861705	34	dbSNP
rs150728160	35	dbSNP
rs762225436	37	dbSNP
rs374874462	39	dbSNP
rs764919705	40	dbSNP
rs571528304	43	dbSNP
rs781657670	45	dbSNP
rs776309910	47	dbSNP
rs1432010093	50	dbSNP
rs1267255103	51	dbSNP
rs185168790	54	dbSNP
rs1476119679	58	dbSNP
rs1323843337	64	dbSNP
rs926322275	66	dbSNP
rs1447226135	68	dbSNP
rs181067791	70	dbSNP
rs1299422019	74	dbSNP
rs980810337	76	dbSNP
rs1001181251	84	dbSNP
rs551648044	85	dbSNP
rs1045408507	86	dbSNP
rs1346846248	95	dbSNP
rs549585549	96	dbSNP
rs1435948505	100	dbSNP
rs1372162716	108	dbSNP
rs748813604	108	dbSNP
rs537909451	111	dbSNP
rs751891156	114	dbSNP
rs115979142	118	dbSNP
rs1344805972	118	dbSNP
rs971907057	118	dbSNP
rs1455008155	119	dbSNP
rs1286989796	123	dbSNP
rs1057221285	124	dbSNP
rs766675368	132	dbSNP
rs1246945680	136	dbSNP
rs1397378718	139	dbSNP
rs987537340	140	dbSNP
rs1377821943	143	dbSNP
rs1477587949	151	dbSNP
rs141159605	158	dbSNP
rs1410077609	160	dbSNP
rs1457756205	161	dbSNP
rs1319801703	164	dbSNP
rs1433157068	165	dbSNP
rs527339828	168	dbSNP
rs1000600303	173	dbSNP
rs1231532042	173	dbSNP
rs1281580959	174	dbSNP
rs942029566	176	dbSNP
rs1228396958	177	dbSNP
rs1197691562	181	dbSNP
rs1326519541	184	dbSNP
rs1267653941	189	dbSNP
rs560203418	190	dbSNP
rs910780651	191	dbSNP
rs1450620147	192	dbSNP
rs904806433	196	dbSNP
rs1234460456	208	dbSNP
rs1472777676	211	dbSNP
rs867520426	212	dbSNP
rs548064628	219	dbSNP
rs867209707	227	dbSNP
rs1409594836	228	dbSNP
rs3802484	233	dbSNP
rs923463630	234	dbSNP
rs758497228	238	dbSNP
rs1211285109	243	dbSNP
rs1349813540	244	dbSNP
rs532878759	247	dbSNP
rs1163843057	257	dbSNP
rs111252342	258	dbSNP
rs1032057652	259	dbSNP
rs1057291951	262	dbSNP
rs1233429418	265	dbSNP
rs369371828	266	dbSNP
rs939121828	278	dbSNP
rs1287027190	281	dbSNP
rs1357870497	282	dbSNP
rs904900997	284	dbSNP
rs1308499580	289	dbSNP
rs1000494919	290	dbSNP
rs969476018	291	dbSNP
rs1276305943	292	dbSNP
rs1483273007	293	dbSNP
rs763177864	295	dbSNP
rs1476111851	302	dbSNP
rs1013946553	310	dbSNP
rs1313140223	314	dbSNP
rs1456432443	316	dbSNP
rs543690233	319	dbSNP
rs1169922091	320	dbSNP
rs896318397	331	dbSNP
rs576458723	336	dbSNP
rs1306490214	338	dbSNP
rs1377815081	340	dbSNP
rs561271394	342	dbSNP
rs146031376	344	dbSNP
rs1339415464	345	dbSNP
rs1465969251	347	dbSNP
rs994010587	347	dbSNP
rs199609109	348	dbSNP
rs940591512	348	dbSNP
rs1183995782	349	dbSNP
rs1417437200	350	dbSNP
rs1342346560	352	dbSNP
rs1251292184	356	dbSNP
rs201387624	356	dbSNP
rs386732990	356	dbSNP
rs750612682	360	dbSNP
rs1188891191	364	dbSNP
rs897873318	368	dbSNP
rs200262967	370	dbSNP
rs773480438	370	dbSNP
rs1038209422	379	dbSNP
rs1277916158	382	dbSNP
rs1255362100	394	dbSNP
rs1422162184	400	dbSNP
rs1199717688	403	dbSNP
rs987233183	408	dbSNP
rs1391025107	410	dbSNP
rs765561685	414	dbSNP
rs528668543	419	dbSNP
rs1377691729	421	dbSNP
rs953526593	426	dbSNP
rs1400640200	436	dbSNP
rs566269149	445	dbSNP
rs116520407	446	dbSNP
rs1365272523	462	dbSNP
rs1227296537	464	dbSNP
rs1236197271	468	dbSNP
rs910571718	479	dbSNP
rs1313792348	482	dbSNP
rs1233646410	485	dbSNP
rs1050680430	488	dbSNP
rs1482197068	493	dbSNP
rs572067989	494	dbSNP
rs1210687997	495	dbSNP
rs933612744	496	dbSNP
rs761967661	507	dbSNP
rs1293417975	513	dbSNP
rs1010505377	514	dbSNP
rs896035685	515	dbSNP
rs1434146364	516	dbSNP
rs549646300	518	dbSNP
rs142522942	519	dbSNP
rs988232513	523	dbSNP
rs112544346	527	dbSNP
rs956869467	529	dbSNP
rs1414263745	531	dbSNP
rs925030477	538	dbSNP
rs979622116	539	dbSNP
rs969063969	540	dbSNP
rs1388785613	547	dbSNP
rs1433685923	548	dbSNP
rs1318154027	552	dbSNP
rs760722082	554	dbSNP
rs1045202100	555	dbSNP
rs1013503092	559	dbSNP
rs1300269084	562	dbSNP
rs538514311	567	dbSNP
rs138071995	578	dbSNP
rs1230226768	581	dbSNP
rs556304933	590	dbSNP
rs898855742	593	dbSNP
rs143709259	597	dbSNP
rs1025571039	603	dbSNP
rs1035960301	604	dbSNP
rs567346647	608	dbSNP
rs1165827884	612	dbSNP
rs911773008	618	dbSNP
rs1488896528	619	dbSNP
rs1476300893	620	dbSNP
rs1265789624	625	dbSNP
rs898344513	634	dbSNP
rs1473757895	635	dbSNP
rs1160619580	644	dbSNP
rs775310539	649	dbSNP
rs1186534905	653	dbSNP
rs1037777121	656	dbSNP
rs1456278645	656	dbSNP
rs771780825	661	dbSNP
rs1006739732	669	dbSNP
rs1458150211	670	dbSNP
rs889118666	673	dbSNP
rs538953590	680	dbSNP
rs933329189	687	dbSNP
rs1203284613	691	dbSNP
rs1292188394	698	dbSNP
rs1328481446	699	dbSNP
rs1273258102	701	dbSNP
rs111808412	709	dbSNP
rs1234610921	712	dbSNP
rs1332124792	721	dbSNP
rs902062526	725	dbSNP
rs1484254976	731	dbSNP
rs1183298966	734	dbSNP
rs1283267353	735	dbSNP
rs1746332	738	dbSNP
rs1184099662	742	dbSNP
rs1415420696	745	dbSNP
rs935490812	746	dbSNP
rs913243774	749	dbSNP
rs1363284259	751	dbSNP
rs1431400764	753	dbSNP
rs925468874	754	dbSNP
rs566448464	755	dbSNP
rs33959301	757	dbSNP
rs979204451	757	dbSNP
rs35196790	758	dbSNP
rs1285847087	759	dbSNP
rs1320733067	763	dbSNP
rs1227765046	771	dbSNP
rs1381015254	778	dbSNP
rs1344504728	783	dbSNP
rs947832061	785	dbSNP
rs916341648	787	dbSNP
rs1388619490	789	dbSNP
rs991784955	809	dbSNP
rs960379404	815	dbSNP
rs149385091	817	dbSNP
rs1383013050	820	dbSNP
rs1451006057	822	dbSNP
rs1001740411	833	dbSNP
rs1389368216	838	dbSNP
rs1463109832	855	dbSNP
rs972673312	856	dbSNP
rs962555829	865	dbSNP
rs1162871273	866	dbSNP
rs1447397650	871	dbSNP
rs75864863	871	dbSNP
rs1475889887	872	dbSNP
rs1367430507	874	dbSNP
rs1374580690	875	dbSNP
rs1006296750	876	dbSNP
rs78593898	882	dbSNP
rs778428462	884	dbSNP
rs1029034884	886	dbSNP
rs997446080	890	dbSNP
rs901882809	891	dbSNP
rs770675645	899	dbSNP
rs1208617783	915	dbSNP
rs1035991016	927	dbSNP
rs1466766802	928	dbSNP
rs1288586882	929	dbSNP
rs1004495860	930	dbSNP
rs890399255	932	dbSNP
rs1052095694	933	dbSNP
rs1214763880	934	dbSNP
rs931892938	936	dbSNP
rs1425803661	937	dbSNP
rs867900017	940	dbSNP
rs1341113954	942	dbSNP
rs935626404	946	dbSNP
rs1174181743	947	dbSNP
rs1390979689	949	dbSNP
rs1435879911	963	dbSNP
rs1320176578	965	dbSNP
rs1324837129	971	dbSNP
rs1439063263	974	dbSNP
rs904014705	979	dbSNP
rs1344746488	991	dbSNP
rs1042861490	996	dbSNP
rs1044249991	997	dbSNP
rs947454656	998	dbSNP
rs1208768584	1008	dbSNP
rs948249111	1012	dbSNP
rs1444864706	1013	dbSNP
rs916293056	1018	dbSNP
rs138818105	1030	dbSNP
rs1247140358	1034	dbSNP
rs1443821874	1037	dbSNP
rs1477559092	1041	dbSNP
rs1197252664	1063	dbSNP
rs1414035871	1066	dbSNP
rs1457837392	1067	dbSNP
rs991922186	1070	dbSNP
rs1407356211	1073	dbSNP
rs1324413158	1076	dbSNP
rs1323060429	1081	dbSNP
rs960016066	1082	dbSNP
rs1398926722	1090	dbSNP
rs1165872496	1092	dbSNP
rs748775705	1093	dbSNP
rs918279226	1099	dbSNP
rs972642081	1100	dbSNP
rs1347919002	1105	dbSNP
rs1211313511	1109	dbSNP
rs1290122863	1111	dbSNP
rs1395556260	1116	dbSNP
rs1195401580	1117	dbSNP
rs962983524	1126	dbSNP
rs970308913	1129	dbSNP
rs550345435	1135	dbSNP
rs1181354883	1142	dbSNP
rs985656266	1154	dbSNP
rs953920035	1163	dbSNP
rs1029003807	1164	dbSNP
rs79572277	1167	dbSNP
rs188670875	1168	dbSNP
rs560825882	1172	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.