ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cgUCGGUCGAGAUGGGGGCGGu 5' :|||:| | :|||||||| Target 5' ggGGCCGGGTGGGCCCCCGCCc 3'	703 - 724	164.00	-30.70
2	miRNA 3' cguCGGUC--GA--GAUGGGGGCGGu 5' ||||| || | :|||||||| Target 5' cccGCCAGGCCTGGCGGCCCCCGCCc 3'	788 - 813	163.00	-28.30
3	miRNA 3' cgUCGGUCGAGAU-----GGGGGCGGu 5' | ||: ||||: |||||||| Target 5' gcACCCGCCTCTGCCGGCCCCCCGCCg 3'	445 - 471	160.00	-31.20

Mutant ID	Mutant Position	Mutant Source
COSN5760357	2	COSMIC
COSN30497585	14	COSMIC
COSN1215225	34	COSMIC
COSN30483268	51	COSMIC
COSN30108306	59	COSMIC
COSN29470834	90	COSMIC
COSN30476574	91	COSMIC
COSN30476614	92	COSMIC
COSN31534415	150	COSMIC
COSN31596022	197	COSMIC
COSN17987902	245	COSMIC
COSN6668547	250	COSMIC
COSN14874519	593	COSMIC
COSN22392956	615	COSMIC
COSN26203714	979	COSMIC
COSN16698422	1046	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1318668924	1	dbSNP
rs1032292909	3	dbSNP
rs1216713712	4	dbSNP
rs1001092837	6	dbSNP
rs201150102	8	dbSNP
rs1206094160	9	dbSNP
rs759715161	10	dbSNP
rs754501506	11	dbSNP
rs753488751	12	dbSNP
rs1044937414	13	dbSNP
rs1328169544	14	dbSNP
rs934190160	14	dbSNP
rs200135539	16	dbSNP
rs1286523347	17	dbSNP
rs1447794587	18	dbSNP
rs370563528	19	dbSNP
rs1167999455	20	dbSNP
rs761204088	22	dbSNP
rs773710056	23	dbSNP
rs200343759	25	dbSNP
rs377328504	26	dbSNP
rs779315260	27	dbSNP
rs1251573122	28	dbSNP
rs924207824	29	dbSNP
rs1200736270	31	dbSNP
rs769468464	33	dbSNP
rs373154952	36	dbSNP
rs1237622643	37	dbSNP
rs780717517	38	dbSNP
rs756727638	41	dbSNP
rs751485306	45	dbSNP
rs1386765776	46	dbSNP
rs1057431464	52	dbSNP
rs565805668	53	dbSNP
rs917516510	54	dbSNP
rs1322361334	55	dbSNP
rs992186024	56	dbSNP
rs372894571	59	dbSNP
rs547033236	60	dbSNP
rs532144458	63	dbSNP
rs919490610	68	dbSNP
rs973704995	71	dbSNP
rs942305744	72	dbSNP
rs779183488	75	dbSNP
rs1015383201	80	dbSNP
rs986272779	84	dbSNP
rs954423832	85	dbSNP
rs1030122604	86	dbSNP
rs1203638577	87	dbSNP
rs571442300	91	dbSNP
rs956426401	93	dbSNP
rs1271160073	94	dbSNP
rs550095241	95	dbSNP
rs1366060571	98	dbSNP
rs996741842	101	dbSNP
rs531383419	103	dbSNP
rs899748265	104	dbSNP
rs1032653044	107	dbSNP
rs1000757724	111	dbSNP
rs1303224115	116	dbSNP
rs1369938434	118	dbSNP
rs1294486927	121	dbSNP
rs754951280	123	dbSNP
rs1336091554	125	dbSNP
rs1352349242	127	dbSNP
rs905123281	134	dbSNP
rs1361681316	137	dbSNP
rs1246887785	145	dbSNP
rs1429784722	150	dbSNP
rs1273989196	162	dbSNP
rs935647653	163	dbSNP
rs1198038375	165	dbSNP
rs1393927790	172	dbSNP
rs1023438978	173	dbSNP
rs1041825962	182	dbSNP
rs949822766	185	dbSNP
rs1455527253	186	dbSNP
rs917446032	189	dbSNP
rs1163721136	192	dbSNP
rs1176351419	193	dbSNP
rs1388983969	196	dbSNP
rs1460434755	198	dbSNP
rs991654917	199	dbSNP
rs748997717	202	dbSNP
rs1395166689	205	dbSNP
rs1334439341	206	dbSNP
rs139054922	208	dbSNP
rs1453624589	210	dbSNP
rs1297010979	212	dbSNP
rs1340089570	212	dbSNP
rs1206854907	215	dbSNP
rs1230289092	220	dbSNP
rs542428395	225	dbSNP
rs1252335576	228	dbSNP
rs1343201224	235	dbSNP
rs1344920113	240	dbSNP
rs1199158568	242	dbSNP
rs1245334919	247	dbSNP
rs1462410412	248	dbSNP
rs530106667	250	dbSNP
rs755632078	251	dbSNP
rs895884061	255	dbSNP
rs946952718	260	dbSNP
rs1302606218	261	dbSNP
rs1465469906	262	dbSNP
rs1174054149	263	dbSNP
rs1057247679	265	dbSNP
rs1320083601	267	dbSNP
rs1359971322	271	dbSNP
rs1384063473	272	dbSNP
rs1296369707	274	dbSNP
rs929281391	281	dbSNP
rs897802211	286	dbSNP
rs1265427339	287	dbSNP
rs1300315074	287	dbSNP
rs973404719	288	dbSNP
rs1203923999	290	dbSNP
rs1267019261	291	dbSNP
rs1410037449	293	dbSNP
rs1206433023	294	dbSNP
rs1261784515	306	dbSNP
rs754166589	307	dbSNP
rs1197137986	310	dbSNP
rs1431189873	311	dbSNP
rs1374542021	317	dbSNP
rs1343714067	318	dbSNP
rs1455522815	324	dbSNP
rs1014916351	329	dbSNP
rs1436056847	330	dbSNP
rs1390060486	333	dbSNP
rs1378083139	336	dbSNP
rs915223908	338	dbSNP
rs1346549189	344	dbSNP
rs990835499	344	dbSNP
rs1252380311	345	dbSNP
rs1182708293	346	dbSNP
rs1483503856	347	dbSNP
rs559423072	347	dbSNP
rs1314812239	349	dbSNP
rs987997374	350	dbSNP
rs1257086305	355	dbSNP
rs541023782	358	dbSNP
rs1426778068	361	dbSNP
rs779973087	361	dbSNP
rs878896570	361	dbSNP
rs954797436	361	dbSNP
rs1160046069	364	dbSNP
rs959473113	364	dbSNP
rs996607890	364	dbSNP
rs899781175	367	dbSNP
rs1035539536	369	dbSNP
rs1422755320	373	dbSNP
rs1305032425	374	dbSNP
rs1366793076	375	dbSNP
rs1409725870	377	dbSNP
rs1296846846	381	dbSNP
rs1032853867	384	dbSNP
rs1225136531	389	dbSNP
rs1484291801	391	dbSNP
rs1307192497	392	dbSNP
rs1358110668	394	dbSNP
rs999828713	396	dbSNP
rs1293294430	398	dbSNP
rs1360125023	401	dbSNP
rs1203686967	405	dbSNP
rs1238926023	414	dbSNP
rs1230986932	419	dbSNP
rs1481461543	420	dbSNP
rs902881588	423	dbSNP
rs1255823103	430	dbSNP
rs1442991263	440	dbSNP
rs1038120523	442	dbSNP
rs1165601163	443	dbSNP
rs981296177	447	dbSNP
rs942255226	448	dbSNP
rs1385905191	449	dbSNP
rs1315718466	451	dbSNP
rs1041381455	452	dbSNP
rs910808803	459	dbSNP
rs8110864	460	dbSNP
rs1229397429	461	dbSNP
rs577206909	462	dbSNP
rs1050552964	468	dbSNP
rs1449975688	468	dbSNP
rs933526876	471	dbSNP
rs1213932936	479	dbSNP
rs1416046936	480	dbSNP
rs922999821	482	dbSNP
rs1187035926	487	dbSNP
rs1309185027	504	dbSNP
rs987930510	507	dbSNP
rs956676902	508	dbSNP
rs1056384372	512	dbSNP
rs971670944	517	dbSNP
rs565434754	519	dbSNP
rs1025441494	522	dbSNP
rs544120647	528	dbSNP
rs979099199	540	dbSNP
rs1159542958	546	dbSNP
rs1474767366	547	dbSNP
rs1417362421	553	dbSNP
rs576826461	554	dbSNP
rs1023594693	558	dbSNP
rs1013413265	561	dbSNP
rs960595644	562	dbSNP
rs1025918690	565	dbSNP
rs993567825	566	dbSNP
rs1364047036	568	dbSNP
rs994007018	570	dbSNP
rs1215052263	571	dbSNP
rs749991625	572	dbSNP
rs897914624	573	dbSNP
rs1205820845	574	dbSNP
rs1485139123	579	dbSNP
rs1270588269	591	dbSNP
rs1446631709	593	dbSNP
rs1037628634	610	dbSNP
rs1213804259	614	dbSNP
rs1209694804	627	dbSNP
rs1243888308	644	dbSNP
rs1476045859	647	dbSNP
rs1199269100	650	dbSNP
rs1427659603	656	dbSNP
rs1481586942	657	dbSNP
rs1177600262	664	dbSNP
rs940918669	665	dbSNP
rs116796197	666	dbSNP
rs1399739197	667	dbSNP
rs1291864911	671	dbSNP
rs1346411163	684	dbSNP
rs889359179	691	dbSNP
rs1431419225	700	dbSNP
rs1282492218	705	dbSNP
rs987475964	706	dbSNP
rs1246506277	707	dbSNP
rs1050662969	709	dbSNP
rs1029056719	710	dbSNP
rs1222239738	711	dbSNP
rs933464147	714	dbSNP
rs1340759621	716	dbSNP
rs1447599206	716	dbSNP
rs1222410354	719	dbSNP
rs1267465871	720	dbSNP
rs1490981063	720	dbSNP
rs1200666798	721	dbSNP
rs1363548984	721	dbSNP
rs1294229522	722	dbSNP
rs975221015	724	dbSNP
rs1156394403	725	dbSNP
rs963815356	728	dbSNP
rs923438128	729	dbSNP
rs1053044180	741	dbSNP
rs1419701474	743	dbSNP
rs536844317	755	dbSNP
rs1032799837	756	dbSNP
rs1363699404	756	dbSNP
rs1321166994	757	dbSNP
rs1406673410	760	dbSNP
rs1303568596	761	dbSNP
rs925026091	764	dbSNP
rs1440310893	766	dbSNP
rs1391979508	769	dbSNP
rs898430693	775	dbSNP
rs1166896776	776	dbSNP
rs1016541158	779	dbSNP
rs1351670680	780	dbSNP
rs1006991514	782	dbSNP
rs1289661214	785	dbSNP
rs979089210	786	dbSNP
rs1223821841	789	dbSNP
rs1250281002	790	dbSNP
rs1013897156	791	dbSNP
rs1179227967	791	dbSNP
rs1234318969	804	dbSNP
rs1439907233	805	dbSNP
rs895557342	807	dbSNP
rs1382809522	810	dbSNP
rs969075706	811	dbSNP
rs1395510565	814	dbSNP
rs916484273	815	dbSNP
rs1330620368	817	dbSNP
rs1408064707	817	dbSNP
rs992053704	819	dbSNP
rs1377984819	820	dbSNP
rs960921128	822	dbSNP
rs937065721	823	dbSNP
rs1025442358	824	dbSNP
rs1313054366	831	dbSNP
rs1385025958	831	dbSNP
rs994411695	833	dbSNP
rs1251181731	835	dbSNP
rs1192486297	837	dbSNP
rs1197988573	838	dbSNP
rs899368786	839	dbSNP
rs1250401629	841	dbSNP
rs1457965602	842	dbSNP
rs1189739235	848	dbSNP
rs962151854	852	dbSNP
rs1182546479	856	dbSNP
rs1016338500	857	dbSNP
rs1445848115	857	dbSNP
rs1006166563	860	dbSNP
rs1187631789	874	dbSNP
rs1271645312	874	dbSNP
rs202160311	876	dbSNP
rs1212075976	879	dbSNP
rs1356530933	882	dbSNP
rs1460379772	883	dbSNP
rs1169464863	890	dbSNP
rs1400703376	896	dbSNP
rs1469506109	898	dbSNP
rs1250429446	899	dbSNP
rs761019516	905	dbSNP
rs1396747798	906	dbSNP
rs1294928855	907	dbSNP
rs940967169	916	dbSNP
rs889137190	919	dbSNP
rs1050402194	926	dbSNP
rs997747885	935	dbSNP
rs908089198	940	dbSNP
rs1202643912	941	dbSNP
rs1323201935	941	dbSNP
rs750966243	942	dbSNP
rs902098286	948	dbSNP
rs35349276	949	dbSNP
rs1260390963	959	dbSNP
rs1426689328	967	dbSNP
rs933299108	967	dbSNP
rs922020341	971	dbSNP
rs1052575742	975	dbSNP
rs935536162	976	dbSNP
rs1479952112	978	dbSNP
rs753210879	988	dbSNP
rs1043520421	989	dbSNP
rs750850809	989	dbSNP
rs1466726090	990	dbSNP
rs947804857	990	dbSNP
rs1288406594	991	dbSNP
rs1382830511	994	dbSNP
rs1384434836	999	dbSNP
rs1299272892	1011	dbSNP
rs572133049	1012	dbSNP
rs1391659393	1018	dbSNP
rs1285556632	1021	dbSNP
rs1167586708	1043	dbSNP
rs958112579	1053	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.