>ZSWIM4|NM_023072|3'UTR
   1 GGGACAGTGGGGTGCGTGGGAGTGGGGATCCCCCTCGCCCCTGCGTCCCCCACCCTTGCTCTCCAATTAGGCCCACGTGG
  81 CATTTCAGTATTATTAAGTCAGGGAAGGAGCCCGGCTGGAGATGGGGGCAGGGGGAGCAGCATCCTGCCACGTGTGTGCC
 161 TGGGAGTCTGTGAGCAAGTGTGCAAGACTCCAGAAGCAGAAGCCATACTGCCACCCAGAAGCCTGGAAGGGGTGTGTGCT
 241 TGGGCTGCTGGTATGACCCCACTTTGGGCACCCCAAAAGCAATAGGCAAACTCCCCTTACCCAGACTGGCTTGGGCTCCA
 321 GGAGGGAGGCTGGGAGGCAGGACTTTCCAGAAATCGACCCTCTAAGTCAATGTAGCACATTTTCCCCCAACCCCACCCTG
 401 GGTGGCCAAGGGTCCTCCCCCACCCCCTACTGCTCAGGGCCCCCCATTATGCCACCTCCAAAGTGGCTGCCCAGCCAGGA
 481 CCATTGGCTCACCCCCACTGAAGGAATTGAAGAGGCCTCAGGCCTCAGCCTCATACCTCACCCCCTTATCCTATCCGGCC
 561 AGGACCCCCATTTCCTTGGGCACTAGGGTCCCAGGGTGGGCTCAGGGTTGGGGAAGTCCTCGCTGCCCTCTCTTCTCACC
 641 TGCTAGTCACTGGACGTACAACTCAGGAACTGAGCGAGGCTCACACTGCCCCTTCCAACCTGGGGTTGGGGGACACAGAC
 721 GCGAGAGAGCCGGAGACCCCCACCCCCGATCTGGAAAGGAAAGAGACCTGTATCTTGGTGTTTTTCTGGCTTTTTCACCC
 801 TTTCGCCTCCTGTCTTTCTGCTTCGCCCCTTCATCTTACTCTGTTTCCATTTTTCCCGGCACTTACCTGCCTTTCTCTCT
 881 GCACCTAAGTCCCTGCCGCCCTCTCTCTTTGGCCATCCTTCCTTTCCCCCACCTTGACCCAGGGAGGAGAAGGGAGAACA
 961 TCACCCCCCCAAGTCACCCCCACATCTTTTCCCTTCTTGTATATTAAGGACAAAATACCACATCATTTTGTAACTTTTTT
1041 TCTATTTATTGAACGGTGTATTACATGTCCTTTTCCTTTTTTTTTTTCAAAGATTGAACAGAAATTTTTCTTTTTAATTT
1121 TATTTTGAGACTGGGGTGCATCTCCAGAGCCACTCACACCCTCAACCTCGTTTCCTCCGC

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' aaGUCGGG---UCGGAGCGGACGAg 5' | |||| :||| |||||| Target 5' cgCTGCCCTCTCTTCTCACCTGCTa 3'	621 - 645	128.00	-22.30
2	miRNA 3' aaGUCGGGUCGGAGCGGACGAg 5' || ||| ::|||||| || Target 5' ttCA-CCC--TTTCGCCTCCTg 3'	794 - 812	127.00	-16.90
3	miRNA 3' aagucggguCGGAGCGGACGAg 5' ||:| | ||||| Target 5' ggggtgtgtGCTTGGGCTGCTg 3'	229 - 250	121.00	-12.00

Mutant ID	Mutant Position	Mutant Source
COSN20047707	12	COSMIC
COSN30525564	17	COSMIC
COSN30515555	22	COSMIC
COSN28886231	38	COSMIC
COSN27006759	45	COSMIC
COSN30130004	46	COSMIC
COSN30540110	102	COSMIC
COSN30451128	108	COSMIC
COSN30451156	109	COSMIC
COSN30158031	115	COSMIC
COSN31570054	125	COSMIC
COSN31545197	130	COSMIC
COSN24387483	134	COSMIC
COSN1211191	227	COSMIC
COSN1760790	314	COSMIC
COSN28753637	358	COSMIC
COSN21445554	480	COSMIC
COSN31533712	640	COSMIC
COSN30174038	665	COSMIC
COSN28750796	724	COSMIC
COSN8343733	866	COSMIC
COSN21445106	1097	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1485518180	6	dbSNP
rs202197140	9	dbSNP
rs748985554	11	dbSNP
rs144297960	12	dbSNP
rs1182579529	16	dbSNP
rs1031921919	17	dbSNP
rs1380540592	18	dbSNP
rs778919819	26	dbSNP
rs1351433342	29	dbSNP
rs748085568	31	dbSNP
rs201378218	36	dbSNP
rs1362514454	37	dbSNP
rs114355642	38	dbSNP
rs771067856	39	dbSNP
rs1404435222	44	dbSNP
rs374641062	45	dbSNP
rs747778923	46	dbSNP
rs1377967648	53	dbSNP
rs1297664644	56	dbSNP
rs1398166770	58	dbSNP
rs1347487324	59	dbSNP
rs1160418680	74	dbSNP
rs1205542332	78	dbSNP
rs963870200	87	dbSNP
rs1423574645	88	dbSNP
rs1179019045	90	dbSNP
rs1481253509	95	dbSNP
rs995097110	103	dbSNP
rs565116449	113	dbSNP
rs762038108	114	dbSNP
rs769338042	117	dbSNP
rs187360665	126	dbSNP
rs1243310570	128	dbSNP
rs982418028	131	dbSNP
rs1441755793	136	dbSNP
rs1223594247	152	dbSNP
rs1253082629	154	dbSNP
rs1324553488	159	dbSNP
rs1277752099	169	dbSNP
rs1216909483	179	dbSNP
rs767782019	185	dbSNP
rs1161619344	194	dbSNP
rs1364226610	196	dbSNP
rs1438493923	199	dbSNP
rs912202023	203	dbSNP
rs965181859	206	dbSNP
rs975567191	224	dbSNP
rs541355894	233	dbSNP
rs559081432	234	dbSNP
rs1381826168	261	dbSNP
rs967727292	272	dbSNP
rs1429589942	273	dbSNP
rs1169563632	288	dbSNP
rs1426345391	293	dbSNP
rs1421094647	296	dbSNP
rs1472427902	297	dbSNP
rs1241135037	301	dbSNP
rs975145934	308	dbSNP
rs921142103	311	dbSNP
rs773697514	314	dbSNP
rs1159569155	326	dbSNP
rs1257158339	329	dbSNP
rs1197043122	340	dbSNP
rs529642735	341	dbSNP
rs921047923	344	dbSNP
rs761017465	348	dbSNP
rs766908004	354	dbSNP
rs924502757	356	dbSNP
rs987040565	357	dbSNP
rs3745453	358	dbSNP
rs1317724235	360	dbSNP
rs879829683	363	dbSNP
rs563057098	364	dbSNP
rs77843146	371	dbSNP
rs1240008178	385	dbSNP
rs1013035315	386	dbSNP
rs1329709223	395	dbSNP
rs1283391702	399	dbSNP
rs367816771	400	dbSNP
rs1170547350	402	dbSNP
rs1422680586	403	dbSNP
rs1229984669	417	dbSNP
rs1273793867	419	dbSNP
rs1457240783	422	dbSNP
rs762346915	422	dbSNP
rs1196288006	423	dbSNP
rs1239456011	427	dbSNP
rs1438062923	429	dbSNP
rs1186320433	430	dbSNP
rs1368285808	435	dbSNP
rs1471986146	436	dbSNP
rs60965837	440	dbSNP
rs994982409	444	dbSNP
rs1294011973	447	dbSNP
rs765911541	457	dbSNP
rs571847577	461	dbSNP
rs1376804809	462	dbSNP
rs1307537629	464	dbSNP
rs1419257031	470	dbSNP
rs1375105399	471	dbSNP
rs1465558059	472	dbSNP
rs1457068691	475	dbSNP
rs1168687175	488	dbSNP
rs948753282	493	dbSNP
rs1044453380	505	dbSNP
rs372024000	508	dbSNP
rs1164924731	512	dbSNP
rs1425299644	513	dbSNP
rs1392184155	517	dbSNP
rs886722583	520	dbSNP
rs548117320	542	dbSNP
rs534539248	544	dbSNP
rs1009135252	546	dbSNP
rs1190875564	553	dbSNP
rs1032267484	558	dbSNP
rs965409420	559	dbSNP
rs1223232887	579	dbSNP
rs1019806572	593	dbSNP
rs892022511	594	dbSNP
rs1317942876	597	dbSNP
rs968535631	602	dbSNP
rs978593503	606	dbSNP
rs73922778	608	dbSNP
rs1304439048	612	dbSNP
rs1021913415	616	dbSNP
rs934615408	617	dbSNP
rs967697585	622	dbSNP
rs975114941	623	dbSNP
rs1455260162	627	dbSNP
rs1364221927	633	dbSNP
rs1175085460	636	dbSNP
rs1479581323	638	dbSNP
rs555071317	652	dbSNP
rs1028036561	656	dbSNP
rs955117125	657	dbSNP
rs986621900	660	dbSNP
rs1207461213	664	dbSNP
rs576584059	673	dbSNP
rs537585502	675	dbSNP
rs111392178	676	dbSNP
rs374634340	677	dbSNP
rs764744145	685	dbSNP
rs540919614	686	dbSNP
rs1223709875	687	dbSNP
rs1339334806	690	dbSNP
rs1253700081	701	dbSNP
rs1383915934	702	dbSNP
rs74901950	704	dbSNP
rs948722316	711	dbSNP
rs1190599231	713	dbSNP
rs1408611940	720	dbSNP
rs10418969	721	dbSNP
rs60981777	722	dbSNP
rs996885749	723	dbSNP
rs12610873	724	dbSNP
rs530507617	726	dbSNP
rs545926714	731	dbSNP
rs1462607606	732	dbSNP
rs978477143	733	dbSNP
rs533132592	738	dbSNP
rs891944367	740	dbSNP
rs1338334668	747	dbSNP
rs372031025	748	dbSNP
rs1228897764	749	dbSNP
rs1326976221	753	dbSNP
rs1314243218	763	dbSNP
rs1021880953	767	dbSNP
rs1372398939	769	dbSNP
rs955836494	776	dbSNP
rs1406433145	802	dbSNP
rs900757987	805	dbSNP
rs996479998	806	dbSNP
rs191007071	810	dbSNP
rs1279963675	822	dbSNP
rs777277215	825	dbSNP
rs532744585	826	dbSNP
rs1446956054	827	dbSNP
rs1231939207	834	dbSNP
rs1244202210	835	dbSNP
rs1253735563	839	dbSNP
rs1056882	841	dbSNP
rs986938016	855	dbSNP
rs948763573	858	dbSNP
rs757088019	859	dbSNP
rs1211080935	863	dbSNP
rs1162575282	865	dbSNP
rs151124535	865	dbSNP
rs1243760711	869	dbSNP
rs1384903512	880	dbSNP
rs920578448	882	dbSNP
rs1441141137	885	dbSNP
rs566393148	898	dbSNP
rs182491944	899	dbSNP
rs961496857	903	dbSNP
rs973918915	904	dbSNP
rs1047836662	906	dbSNP
rs1161739374	910	dbSNP
rs1427114006	913	dbSNP
rs1386340176	926	dbSNP
rs1056883	932	dbSNP
rs548587070	943	dbSNP
rs1468886884	954	dbSNP
rs1429627848	957	dbSNP
rs373673610	964	dbSNP
rs554631812	964	dbSNP
rs1183600366	966	dbSNP
rs570104287	970	dbSNP
rs1455750278	971	dbSNP
rs1292349753	972	dbSNP
rs367674027	975	dbSNP
rs1348329044	977	dbSNP
rs1359689842	977	dbSNP
rs1281054504	980	dbSNP
rs1240107400	983	dbSNP
rs529165331	986	dbSNP
rs1056884	997	dbSNP
rs1290379623	1001	dbSNP
rs1301383990	1009	dbSNP
rs1406793177	1010	dbSNP
rs969959308	1015	dbSNP
rs1386129854	1019	dbSNP
rs1399441591	1024	dbSNP
rs1298569907	1025	dbSNP
rs1410693371	1035	dbSNP
rs1332250590	1048	dbSNP
rs781309559	1055	dbSNP
rs868288594	1056	dbSNP
rs1196683208	1058	dbSNP
rs1490687091	1061	dbSNP
rs1268944703	1064	dbSNP
rs1309925995	1077	dbSNP
rs996424684	1077	dbSNP
rs1224072707	1078	dbSNP
rs1351014188	1080	dbSNP
rs745754275	1082	dbSNP
rs186816048	1088	dbSNP
rs1214994629	1089	dbSNP
rs1346284136	1090	dbSNP
rs1269661308	1093	dbSNP
rs1429322939	1095	dbSNP
rs1347395883	1097	dbSNP
rs904187710	1100	dbSNP
rs1469570171	1111	dbSNP
rs1404942835	1112	dbSNP
rs1262854044	1116	dbSNP
rs1486489108	1118	dbSNP
rs1209697491	1123	dbSNP
rs1481202801	1128	dbSNP
rs570510334	1131	dbSNP
rs554318919	1137	dbSNP
rs1056887	1138	dbSNP
rs1185359664	1145	dbSNP
rs1474392183	1150	dbSNP
rs1469843634	1151	dbSNP
rs1194315521	1158	dbSNP
rs1243724557	1159	dbSNP
rs1216273275	1162	dbSNP
rs1460735158	1166	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.