ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ugacaGACAGAGUCUUGGGGuc 5' ||| || |||||: Target 5' atgacCTGCATCCTAACCCTtg 3'	68 - 89	117.00	-11.60
2	miRNA 3' ugACAGACAGAGUCUUG-GGGUc 5' || | | : ||:|| |||| Target 5' agTGCCAGGTGGAGGACTCCCAt 3'	103 - 125	108.00	-10.10
3	miRNA 3' ugacagacAGAGUCUUGGGGUc 5' ||| : ||::|:| Target 5' gcaagaaaTCTGGAAATTCTAt 3'	21 - 42	102.00	-5.59

Mutant ID	Mutant Position	Mutant Source
rs369509310	12	dbSNP
rs1467080043	14	dbSNP
rs759544335	14	dbSNP
rs765184423	16	dbSNP
rs1453722104	32	dbSNP
rs775371026	34	dbSNP
rs1195559336	42	dbSNP
rs768698797	46	dbSNP
rs1205089754	52	dbSNP
rs776755593	80	dbSNP
rs1000459783	89	dbSNP
rs1249913076	89	dbSNP
rs1001323715	98	dbSNP
rs1018011425	99	dbSNP
rs1254621016	111	dbSNP
rs1234065869	134	dbSNP
rs1339204170	138	dbSNP
rs1205959815	153	dbSNP
rs899595610	156	dbSNP
rs1482691132	159	dbSNP
rs1333412579	161	dbSNP
rs761917441	164	dbSNP
rs13340756	165	dbSNP
rs1398269432	176	dbSNP
rs1310814785	183	dbSNP
rs773358306	191	dbSNP
rs1052450359	211	dbSNP
rs140575080	230	dbSNP
rs1468544604	248	dbSNP
rs1363288635	268	dbSNP
rs1423055189	269	dbSNP
rs1218445141	275	dbSNP
rs375237575	282	dbSNP
rs188516894	289	dbSNP
rs1172555092	294	dbSNP
rs1373866483	303	dbSNP
rs751909555	320	dbSNP
rs1254391226	329	dbSNP
rs1465730631	339	dbSNP
rs1486182248	343	dbSNP
rs542524482	344	dbSNP
rs1282920028	348	dbSNP
rs1227524824	349	dbSNP
rs1278442460	358	dbSNP
rs1025259422	361	dbSNP
rs1377028474	371	dbSNP
rs1337141769	397	dbSNP
rs1291007987	408	dbSNP
rs1413791982	417	dbSNP
rs1375772241	425	dbSNP
rs969610486	428	dbSNP
rs1398875880	431	dbSNP
rs954924412	456	dbSNP
rs181059913	457	dbSNP
rs1015169719	467	dbSNP
rs767981484	475	dbSNP
rs1415806108	484	dbSNP
rs1215829821	490	dbSNP
rs1475667295	491	dbSNP
rs1260038595	498	dbSNP
rs963560699	513	dbSNP
rs144795909	514	dbSNP
rs528098972	530	dbSNP
rs780432044	533	dbSNP
rs1311317065	537	dbSNP
rs1352165703	548	dbSNP
rs937424730	549	dbSNP
rs2213514	558	dbSNP
rs374951167	559	dbSNP
rs755141815	564	dbSNP
rs1041636990	566	dbSNP
rs1294863630	580	dbSNP
rs951836554	581	dbSNP
rs1325660538	589	dbSNP
rs984907496	596	dbSNP
rs904467670	624	dbSNP
rs781422730	625	dbSNP
rs1402383141	636	dbSNP
rs1211990211	688	dbSNP
rs1392308920	697	dbSNP
rs1164380016	700	dbSNP
rs1462161743	705	dbSNP
rs1263124848	727	dbSNP
rs1420347505	729	dbSNP
rs778227916	733	dbSNP
rs935822206	734	dbSNP
rs1039963204	737	dbSNP
rs1487558690	750	dbSNP
rs1243087214	756	dbSNP
rs899544018	757	dbSNP
rs923232865	761	dbSNP
rs1250591995	765	dbSNP
rs1262503691	766	dbSNP
rs1335046698	779	dbSNP
rs1274604996	789	dbSNP
rs186557162	790	dbSNP
rs998353935	797	dbSNP
rs868786555	814	dbSNP
rs1397077856	819	dbSNP
rs1368785971	835	dbSNP
rs1325270419	844	dbSNP
rs1441799653	849	dbSNP
rs1396812748	850	dbSNP
rs1168553444	855	dbSNP
rs886772771	857	dbSNP
rs913364117	861	dbSNP
rs1420652005	864	dbSNP
rs1435128445	880	dbSNP
rs1453083760	887	dbSNP
rs1194822421	888	dbSNP
rs757979836	888	dbSNP
rs866110170	888	dbSNP
rs1201938831	889	dbSNP
rs1157767173	907	dbSNP
rs1358553616	908	dbSNP
rs1273445406	950	dbSNP
rs1003858718	952	dbSNP
rs779908637	954	dbSNP
rs1345614445	957	dbSNP
rs368820361	962	dbSNP
rs1302527425	965	dbSNP
rs1234971134	966	dbSNP
rs1015100631	978	dbSNP
rs963984278	982	dbSNP
rs925069947	990	dbSNP
rs936788341	995	dbSNP
rs7879097	999	dbSNP
rs1384755234	1000	dbSNP
rs1394884614	1001	dbSNP
rs768608836	1002	dbSNP
rs1455975796	1022	dbSNP
rs1034938596	1035	dbSNP
rs75981565	1051	dbSNP
rs776594946	1058	dbSNP
rs1426650770	1059	dbSNP
rs1175045267	1068	dbSNP
rs958934312	1068	dbSNP
rs1469283680	1071	dbSNP
rs1233153450	1085	dbSNP
rs946197492	1122	dbSNP
rs1305216526	1130	dbSNP
rs190662039	1133	dbSNP
rs769778353	1142	dbSNP
rs917360789	1146	dbSNP
rs949388752	1169	dbSNP
rs1046428217	1171	dbSNP
rs905476867	1183	dbSNP
rs967356813	1202	dbSNP
rs977434012	1218	dbSNP
rs769931167	1230	dbSNP
rs935921208	1244	dbSNP
rs1039507885	1247	dbSNP
rs1313934535	1257	dbSNP
rs1437123228	1264	dbSNP
rs888587763	1319	dbSNP
rs1005980950	1320	dbSNP
rs1408119245	1346	dbSNP
rs1417952851	1346	dbSNP
rs1183814286	1355	dbSNP
rs1015113092	1358	dbSNP
rs921089282	1358	dbSNP
rs1188438714	1365	dbSNP
rs961769113	1376	dbSNP
rs1258841100	1378	dbSNP
rs933879802	1385	dbSNP
rs182900448	1394	dbSNP
rs1242820823	1399	dbSNP
rs1270102302	1408	dbSNP
rs1488216478	1414	dbSNP
rs1029921437	1416	dbSNP
rs953300344	1429	dbSNP
rs749315156	1430	dbSNP
rs1003809651	1437	dbSNP
rs1288757616	1445	dbSNP
rs1177848381	1448	dbSNP
rs1454767273	1450	dbSNP
rs1191064445	1463	dbSNP
rs925154773	1469	dbSNP
rs1160821626	1474	dbSNP
rs763153530	1494	dbSNP
rs1416778957	1495	dbSNP
rs936531148	1497	dbSNP
rs1425292245	1518	dbSNP
rs1259399511	1531	dbSNP
rs1185796595	1532	dbSNP
rs1402423744	1537	dbSNP
rs1249980188	1551	dbSNP
rs1159701540	1575	dbSNP
rs1349186486	1577	dbSNP
rs1455951538	1581	dbSNP
rs899380731	1583	dbSNP
rs73250631	1585	dbSNP
rs766568384	1585	dbSNP
rs914012255	1585	dbSNP
rs760881879	1600	dbSNP
rs1295327728	1618	dbSNP
rs1371370159	1624	dbSNP
rs1034644829	1626	dbSNP
rs1321943006	1631	dbSNP
rs1438017930	1637	dbSNP
rs1309055773	1651	dbSNP
rs143596333	1652	dbSNP
rs1459130207	1652	dbSNP
rs1225792261	1658	dbSNP
rs1014458597	1671	dbSNP
rs1169560802	1679	dbSNP
rs761231066	1679	dbSNP
rs1429020214	1701	dbSNP
rs1046440782	1702	dbSNP
rs1468061929	1729	dbSNP
rs1253709580	1732	dbSNP
rs770818986	1738	dbSNP
rs767821484	1740	dbSNP
rs977381758	1744	dbSNP
rs1469119196	1745	dbSNP
rs5967201	1745	dbSNP
rs1198223764	1746	dbSNP
rs925956833	1753	dbSNP
rs1301827738	1757	dbSNP
rs1471055706	1759	dbSNP
rs938261009	1761	dbSNP
rs1049091896	1767	dbSNP
rs1412991319	1781	dbSNP
rs1421910799	1782	dbSNP
rs957429881	1787	dbSNP
rs1323708580	1788	dbSNP
rs1388144340	1803	dbSNP
rs1166549161	1814	dbSNP
rs888689816	1818	dbSNP
rs1004828870	1820	dbSNP
rs975734034	1824	dbSNP
rs1464817863	1843	dbSNP
rs1171613717	1859	dbSNP
rs1453742775	1862	dbSNP
rs921215643	1869	dbSNP
rs933813822	1870	dbSNP
rs1417529642	1871	dbSNP
rs113280965	1874	dbSNP
rs753119361	1885	dbSNP
rs1484200926	1894	dbSNP
rs1248991749	1910	dbSNP
rs908260135	1915	dbSNP
rs939781236	1924	dbSNP
rs985802757	1935	dbSNP
rs1444834441	1952	dbSNP
rs187121793	1956	dbSNP
rs1038156296	1957	dbSNP
rs1280436205	1967	dbSNP
rs898150114	1978	dbSNP
rs1333502874	1983	dbSNP
rs1305802576	1985	dbSNP
rs1410060439	1994	dbSNP
rs1231985409	2004	dbSNP
rs1352042329	2028	dbSNP
rs1032695800	2034	dbSNP
rs1295253529	2045	dbSNP
rs1434025475	2045	dbSNP
rs1001898965	2075	dbSNP
rs764727889	2078	dbSNP
rs894739409	2080	dbSNP
rs1014409518	2082	dbSNP
rs759428020	2090	dbSNP
rs1183089933	2099	dbSNP
rs967421758	2110	dbSNP
rs998932668	2114	dbSNP
rs1187725377	2117	dbSNP
rs1204956940	2126	dbSNP
rs1486271261	2145	dbSNP
rs1033140061	2149	dbSNP
rs1203929270	2171	dbSNP
rs1236670379	2174	dbSNP
rs868636668	2176	dbSNP
rs957584838	2185	dbSNP
rs1228612410	2195	dbSNP
rs975470283	2196	dbSNP
rs1028631682	2198	dbSNP
rs1413885257	2199	dbSNP
rs955292769	2202	dbSNP
rs1418439358	2211	dbSNP
rs986560873	2216	dbSNP
rs1336015190	2217	dbSNP
rs908373509	2221	dbSNP
rs1384330225	2225	dbSNP
rs749885951	2227	dbSNP
rs1160083383	2232	dbSNP
rs1458988200	2241	dbSNP
rs112627370	2246	dbSNP
rs1164511995	2256	dbSNP
rs1473308177	2259	dbSNP
rs775289253	2268	dbSNP
rs1191593786	2273	dbSNP
rs1446995724	2276	dbSNP
rs1410178898	2277	dbSNP
rs1246882733	2284	dbSNP
rs1208857158	2289	dbSNP
rs1330750047	2292	dbSNP
rs919716104	2299	dbSNP
rs1232303655	2300	dbSNP
rs1356710825	2309	dbSNP
rs937644055	2311	dbSNP
rs765401301	2328	dbSNP
rs1054816583	2329	dbSNP
rs1413512997	2336	dbSNP
rs1321273836	2340	dbSNP
rs1325825273	2340	dbSNP
rs1437303374	2347	dbSNP
rs1315394440	2351	dbSNP
rs1392416247	2367	dbSNP
rs1325395328	2369	dbSNP
rs779818627	2380	dbSNP
rs894692680	2386	dbSNP
rs751211682	2389	dbSNP
rs141629759	2408	dbSNP
rs903348693	2412	dbSNP
rs1268418759	2422	dbSNP
rs1341810480	2425	dbSNP
rs999059643	2432	dbSNP
rs1166589795	2436	dbSNP
rs1475357729	2437	dbSNP
rs1033500778	2445	dbSNP
rs1391381805	2452	dbSNP
rs1191086022	2453	dbSNP
rs1465804580	2461	dbSNP
rs1250609242	2473	dbSNP
rs1220353498	2497	dbSNP
rs893235891	2523	dbSNP
rs997210017	2527	dbSNP
rs1212637069	2531	dbSNP
rs7062004	2547	dbSNP
rs901326554	2561	dbSNP
rs111821384	2587	dbSNP
rs955406730	2596	dbSNP
rs748045325	2607	dbSNP
rs1437456866	2614	dbSNP
rs1051489957	2630	dbSNP
rs1487884672	2635	dbSNP
rs763721458	2651	dbSNP
rs961182623	2653	dbSNP
rs973945663	2663	dbSNP
rs1392714432	2670	dbSNP
rs1475565243	2673	dbSNP
rs1190168600	2674	dbSNP
rs1478138563	2687	dbSNP
rs1007676543	2694	dbSNP
rs111425079	2703	dbSNP
rs1392833602	2706	dbSNP
rs1275108245	2712	dbSNP
rs1433270655	2722	dbSNP
rs1175853534	2727	dbSNP
rs1345728219	2740	dbSNP
rs777816815	2743	dbSNP
rs1219941738	2756	dbSNP
rs1376595333	2761	dbSNP
rs1369852316	2778	dbSNP
rs1277930679	2781	dbSNP
rs1441764910	2787	dbSNP
rs990858933	2794	dbSNP
rs1351468876	2824	dbSNP
rs373189427	2829	dbSNP
rs1433366289	2830	dbSNP
rs949072538	2835	dbSNP
rs758988598	2840	dbSNP
rs1375425078	2856	dbSNP
rs903467716	2858	dbSNP
rs1316383241	2874	dbSNP
rs1414502703	2883	dbSNP
rs183028366	2903	dbSNP
rs1431524514	2906	dbSNP
rs1472093765	2910	dbSNP
rs1252271055	2936	dbSNP
rs1184132489	2943	dbSNP
rs1484639727	2947	dbSNP
rs958203077	2948	dbSNP
rs1009446742	2949	dbSNP
rs1313962070	2951	dbSNP
rs1054650278	2974	dbSNP
rs1228007685	2975	dbSNP
rs138737369	2976	dbSNP
rs187652717	2978	dbSNP
rs1413269283	2984	dbSNP
rs996931259	2986	dbSNP
rs1378938188	2992	dbSNP
rs1236229045	2995	dbSNP
rs1303869028	3020	dbSNP
rs755338291	3024	dbSNP
rs1360378295	3043	dbSNP
rs1156399166	3061	dbSNP
rs781332722	3068	dbSNP
rs1352973382	3073	dbSNP
rs1203288946	3075	dbSNP
rs926716334	3085	dbSNP
rs1285792769	3086	dbSNP
rs1182359611	3100	dbSNP
rs891105243	3110	dbSNP
rs1008211588	3113	dbSNP
rs1484230951	3115	dbSNP
rs1447108606	3126	dbSNP
rs1208240424	3130	dbSNP
rs959478255	3132	dbSNP
rs1270202623	3153	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.