ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cuAAUUUGUUAAGAGAc 5' | ||:||||||||| Target 5' gtTCAAGCAATTCTCTg 3'	914 - 930	163.00	-16.10
2	miRNA 3' cuAAUUUGUUAAGAGAc 5' |||| |||||||| Target 5' gaTTAA--AATTCTCTt 3'	1490 - 1504	155.00	-8.40
3	miRNA 3' cuAAUUUGUUAAGAGAc 5' ||:|| |||:||| Target 5' atTTGAAACATTTTCTa 3'	178 - 194	139.00	-5.20

Mutant ID	Mutant Position	Mutant Source
1193231	280	ClinVar
COSN26972689	4	COSMIC
COSN28163137	17	COSMIC
COSN26972687	18	COSMIC
COSN2243750	23	COSMIC
COSN17075760	26	COSMIC
COSN31562125	43	COSMIC
COSN30456392	45	COSMIC
COSN26972688	54	COSMIC
COSN14308212	57	COSMIC
COSN31536715	88	COSMIC
COSN31558558	152	COSMIC
COSN30133883	159	COSMIC
COSN4935838	232	COSMIC
COSN32057790	377	COSMIC
COSN31849247	507	COSMIC
COSN32057789	631	COSMIC
COSN22187368	664	COSMIC
COSN20591553	670	COSMIC
COSN22604933	934	COSMIC
COSN2243748	936	COSMIC
COSN2243746	937	COSMIC
COSN27045156	1513	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs749035329	5	dbSNP
rs1168076514	9	dbSNP
rs376536889	17	dbSNP
rs755694531	18	dbSNP
rs1199901706	22	dbSNP
rs1433812561	27	dbSNP
rs1432816865	28	dbSNP
rs368023689	33	dbSNP
rs1318667130	34	dbSNP
rs766927510	37	dbSNP
rs200373494	38	dbSNP
rs1275206126	39	dbSNP
rs550698093	42	dbSNP
rs766932673	44	dbSNP
rs763430580	50	dbSNP
rs1382462943	53	dbSNP
rs953156415	54	dbSNP
rs369608214	56	dbSNP
rs1433444473	57	dbSNP
rs150868543	59	dbSNP
rs1305892196	64	dbSNP
rs142175224	79	dbSNP
rs1254670397	83	dbSNP
rs1015730709	94	dbSNP
rs1383337211	99	dbSNP
rs1367331592	105	dbSNP
rs1324317071	114	dbSNP
rs1205970724	144	dbSNP
rs778062325	146	dbSNP
rs1463607425	156	dbSNP
rs1004317313	158	dbSNP
rs551935774	159	dbSNP
rs1442595728	167	dbSNP
rs1253519190	168	dbSNP
rs1394791394	168	dbSNP
rs1330321370	174	dbSNP
rs1173644712	191	dbSNP
rs528619115	195	dbSNP
rs559556007	197	dbSNP
rs1374359968	202	dbSNP
rs1453943376	202	dbSNP
rs549209509	203	dbSNP
rs529130704	205	dbSNP
rs777618375	206	dbSNP
rs1456236757	211	dbSNP
rs563639217	212	dbSNP
rs1326055816	226	dbSNP
rs312262915	228	dbSNP
rs556387161	243	dbSNP
rs7793861	251	dbSNP
rs577594478	265	dbSNP
rs1330900801	269	dbSNP
rs1253846965	272	dbSNP
rs905195252	275	dbSNP
rs1270250807	279	dbSNP
rs115141387	280	dbSNP
rs949341620	289	dbSNP
rs892365538	293	dbSNP
rs1189412151	297	dbSNP
rs1052292534	302	dbSNP
rs1253205499	316	dbSNP
rs1228721139	322	dbSNP
rs372348151	323	dbSNP
rs1184147062	334	dbSNP
rs1312153346	338	dbSNP
rs540688443	341	dbSNP
rs1456415546	346	dbSNP
rs1164961457	358	dbSNP
rs1396062286	364	dbSNP
rs986283465	367	dbSNP
rs1301640733	369	dbSNP
rs1236927052	373	dbSNP
rs6465348	377	dbSNP
rs767215174	379	dbSNP
rs922941024	384	dbSNP
rs1293922925	402	dbSNP
rs1219552799	403	dbSNP
rs529550688	403	dbSNP
rs1264779640	404	dbSNP
rs1480947495	405	dbSNP
rs1206994622	408	dbSNP
rs1249391536	410	dbSNP
rs1472033217	411	dbSNP
rs1188238154	416	dbSNP
rs1415254563	417	dbSNP
rs1298253256	420	dbSNP
rs965621269	424	dbSNP
rs1163534503	426	dbSNP
rs1368365238	428	dbSNP
rs1373585197	433	dbSNP
rs1430982202	434	dbSNP
rs553102640	436	dbSNP
rs1310183819	437	dbSNP
rs1369108075	441	dbSNP
rs1409367318	443	dbSNP
rs1313708606	449	dbSNP
rs1015784729	454	dbSNP
rs1454529791	464	dbSNP
rs542858380	472	dbSNP
rs34661504	478	dbSNP
rs952878431	478	dbSNP
rs1181346841	480	dbSNP
rs1230552413	485	dbSNP
rs1027114777	497	dbSNP
rs1272144809	497	dbSNP
rs1211419502	498	dbSNP
rs1238834483	508	dbSNP
rs1482091875	510	dbSNP
rs1180729187	513	dbSNP
rs1417573150	530	dbSNP
rs1450573397	539	dbSNP
rs748075593	545	dbSNP
rs1454617015	547	dbSNP
rs1196476879	549	dbSNP
rs1482993794	558	dbSNP
rs761571058	563	dbSNP
rs1361081522	580	dbSNP
rs1213722173	588	dbSNP
rs1363322645	590	dbSNP
rs1446132633	607	dbSNP
rs1302290504	608	dbSNP
rs1002708046	609	dbSNP
rs1295312918	611	dbSNP
rs1341444783	622	dbSNP
rs1234726921	629	dbSNP
rs57122389	631	dbSNP
rs1025414870	641	dbSNP
rs1013575134	655	dbSNP
rs892419528	657	dbSNP
rs958116547	671	dbSNP
rs1337197743	680	dbSNP
rs1052346781	696	dbSNP
rs192719001	697	dbSNP
rs1192098687	700	dbSNP
rs1427723979	701	dbSNP
rs903693290	703	dbSNP
rs1050239367	714	dbSNP
rs1389751832	715	dbSNP
rs1394928113	719	dbSNP
rs1458879607	726	dbSNP
rs1353067255	731	dbSNP
rs931741327	738	dbSNP
rs1437130142	752	dbSNP
rs923025348	757	dbSNP
rs1365228067	762	dbSNP
rs1218495791	771	dbSNP
rs1040471134	775	dbSNP
rs1330834586	778	dbSNP
rs940352911	780	dbSNP
rs1266975770	781	dbSNP
rs1464804086	785	dbSNP
rs11459	792	dbSNP
rs1248753552	801	dbSNP
rs1477243628	811	dbSNP
rs1451832259	812	dbSNP
rs1365732192	813	dbSNP
rs1182848814	814	dbSNP
rs867279541	816	dbSNP
rs868029572	817	dbSNP
rs1325439206	818	dbSNP
rs1473721046	818	dbSNP
rs571588380	818	dbSNP
rs1190619798	819	dbSNP
rs1486286208	820	dbSNP
rs1285703179	821	dbSNP
rs1288792919	821	dbSNP
rs1203782222	822	dbSNP
rs1351906468	830	dbSNP
rs1353355436	836	dbSNP
rs1221261896	837	dbSNP
rs1226214171	837	dbSNP
rs1287097656	837	dbSNP
rs1480495436	837	dbSNP
rs879010642	837	dbSNP
rs1484123945	846	dbSNP
rs1260919528	847	dbSNP
rs553092501	849	dbSNP
rs1473158329	850	dbSNP
rs1165122095	856	dbSNP
rs1411492067	857	dbSNP
rs1404725515	859	dbSNP
rs1332018629	861	dbSNP
rs1294612424	865	dbSNP
rs1415251548	866	dbSNP
rs189164126	870	dbSNP
rs1307024253	875	dbSNP
rs1340763092	881	dbSNP
rs371794707	882	dbSNP
rs1281965977	890	dbSNP
rs1354818586	894	dbSNP
rs1383672469	895	dbSNP
rs1385486295	896	dbSNP
rs1275708913	901	dbSNP
rs549188846	911	dbSNP
rs766560493	912	dbSNP
rs529120368	917	dbSNP
rs1167225835	919	dbSNP
rs920102106	920	dbSNP
rs184666377	923	dbSNP
rs1472392930	924	dbSNP
rs1169148233	928	dbSNP
rs1392023042	932	dbSNP
rs1242025471	934	dbSNP
rs367964957	936	dbSNP
rs1167786409	937	dbSNP
rs1402440039	938	dbSNP
rs312262916	944	dbSNP
rs1331573124	945	dbSNP
rs1262940487	951	dbSNP
rs1339383724	951	dbSNP
rs1212617098	952	dbSNP
rs1298989121	953	dbSNP
rs1344989915	957	dbSNP
rs969901042	964	dbSNP
rs1013629080	968	dbSNP
rs563697409	984	dbSNP
rs1202401008	996	dbSNP
rs956690068	1007	dbSNP
rs1135217	1008	dbSNP
rs1189366306	1010	dbSNP
rs1187640021	1013	dbSNP
rs1249778797	1013	dbSNP
rs1378017012	1013	dbSNP
rs1447632643	1013	dbSNP
rs12673910	1016	dbSNP
rs1316796796	1019	dbSNP
rs1170436295	1020	dbSNP
rs903742883	1021	dbSNP
rs1457284732	1027	dbSNP
rs187286657	1028	dbSNP
rs1367913111	1029	dbSNP
rs1433567607	1032	dbSNP
rs1295220561	1034	dbSNP
rs1332055089	1036	dbSNP
rs1235960030	1039	dbSNP
rs1273293809	1040	dbSNP
rs1323142677	1042	dbSNP
rs1326742021	1046	dbSNP
rs1270114671	1048	dbSNP
rs1463454771	1049	dbSNP
rs996009074	1050	dbSNP
rs901622275	1052	dbSNP
rs1434687029	1053	dbSNP
rs1440391275	1054	dbSNP
rs1196533045	1056	dbSNP
rs1428141074	1059	dbSNP
rs1472727779	1060	dbSNP
rs1160688478	1061	dbSNP
rs1040119422	1064	dbSNP
rs1288782521	1065	dbSNP
rs1328235086	1078	dbSNP
rs1388854985	1083	dbSNP
rs1462004021	1083	dbSNP
rs1299701770	1093	dbSNP
rs542838738	1106	dbSNP
rs185412516	1107	dbSNP
rs1166653539	1108	dbSNP
rs1327151078	1109	dbSNP
rs760596755	1111	dbSNP
rs1183704705	1113	dbSNP
rs868344904	1117	dbSNP
rs312262910	1121	dbSNP
rs557294385	1131	dbSNP
rs1193438481	1135	dbSNP
rs1048682465	1149	dbSNP
rs1466824210	1156	dbSNP
rs1417995435	1158	dbSNP
rs1164911082	1160	dbSNP
rs1369528917	1161	dbSNP
rs1459915972	1169	dbSNP
rs1292105747	1179	dbSNP
rs931584577	1179	dbSNP
rs1410600971	1180	dbSNP
rs1254455143	1181	dbSNP
rs771657181	1182	dbSNP
rs1390871194	1189	dbSNP
rs1214004686	1192	dbSNP
rs1323390676	1192	dbSNP
rs1436708605	1215	dbSNP
rs1270289175	1225	dbSNP
rs759093712	1225	dbSNP
rs1445871458	1227	dbSNP
rs1290686965	1234	dbSNP
rs1342589710	1237	dbSNP
rs780880241	1240	dbSNP
rs1197410735	1241	dbSNP
rs544062295	1244	dbSNP
rs1181883284	1245	dbSNP
rs1257693547	1257	dbSNP
rs1426002006	1258	dbSNP
rs1302349700	1267	dbSNP
rs1385236233	1276	dbSNP
rs1430776224	1277	dbSNP
rs1231780279	1286	dbSNP
rs1352934277	1292	dbSNP
rs1303435153	1300	dbSNP
rs969577521	1300	dbSNP
rs1381449072	1303	dbSNP
rs1394424552	1312	dbSNP
rs1372969049	1313	dbSNP
rs537438608	1313	dbSNP
rs192593756	1315	dbSNP
rs992248889	1322	dbSNP
rs1245831411	1324	dbSNP
rs751270903	1324	dbSNP
rs956744599	1326	dbSNP
rs1031024130	1327	dbSNP
rs1326940355	1328	dbSNP
rs188361781	1329	dbSNP
rs759494067	1335	dbSNP
rs1450665236	1338	dbSNP
rs1190867173	1339	dbSNP
rs1417822141	1340	dbSNP
rs1397866106	1341	dbSNP
rs1409545891	1341	dbSNP
rs1361187261	1346	dbSNP
rs535068396	1351	dbSNP
rs1336216614	1360	dbSNP
rs1028899847	1362	dbSNP
rs770698483	1369	dbSNP
rs1295041193	1377	dbSNP
rs1361022423	1380	dbSNP
rs1219645487	1396	dbSNP
rs1302920065	1405	dbSNP
rs1397334959	1409	dbSNP
rs1193814279	1413	dbSNP
rs1268251170	1414	dbSNP
rs748947706	1415	dbSNP
rs375581416	1417	dbSNP
rs528139567	1417	dbSNP
rs1240925297	1419	dbSNP
rs1475289628	1426	dbSNP
rs1192177890	1427	dbSNP
rs901677507	1432	dbSNP
rs1175749857	1434	dbSNP
rs1230632959	1440	dbSNP
rs1183380716	1441	dbSNP
rs1412798079	1446	dbSNP
rs1458693272	1454	dbSNP
rs1344601421	1455	dbSNP
rs1436909713	1457	dbSNP
rs1437954347	1460	dbSNP
rs1343214341	1461	dbSNP
rs55776212	1461	dbSNP
rs1279487882	1467	dbSNP
rs1222533210	1470	dbSNP
rs1208727500	1478	dbSNP
rs1288368387	1478	dbSNP
rs1040149158	1484	dbSNP
rs1280803524	1485	dbSNP
rs1004602070	1498	dbSNP
rs1222707491	1499	dbSNP
rs1248850080	1502	dbSNP
rs1489901019	1504	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.