>BAK1|NM_001188|3'UTR
   1 CTCCCAAGGGTGCCCTTTGGGGTCCCGGTTCAGACCCCTGCCTGGACTTAAGCGAAGTCTTTGCCTTCTCTGTTCCCTTG
  81 CAGGGGTCCCCCCTCAAGAGTACAGAAGCTTTAGCAAGTGTGCACTCCAGCTTCGGAGGGCCCCTGCGTGGGGGCCAGTC
 161 AGGCTGCAGAGGCACCTCAACATTGCATGGTGCTAGTGGGCCCTCTCTCTGGGCCCAGGGGCTGTGGCCGTCTCCTCCCT
 241 CAGCTCTCTGGGACCTCCTTAGCCCTGTCTGCTAGGCGCTGGGGAGACTGATAACTTGGGGAGGCAAGAGACTGGGAGCC
 321 ACTTCTCCCCAGAAAGTGTTTAACGGTTTTAGCTTTTTATAATACCCTTGTGAGAGCCCATTCCCACCATTCTACCTGAG
 401 GCCAGGACGTCTGGGGTGTGGGGATTGGTGGGTCTATGTTCCCCAGGATTCAGCTATTCTGGAAGATCAGCACCCTAAGA
 481 GATGGGACTAGGACCTGAGCCTGGTCCTGGCCGTCCCTAAGCATGTGTCCCAGGAGCAGGACCTACTAGGAGAGGGGGGC
 561 CAAGGTCCTGCTCAACTCTACCCCTGCTCCCATTCCTCCCTCCGGCCATACTGCCTTTGCAGTTGGACTCTCAGGGATTC
 641 TGGGCTTGGGGTGTGGGGTGGGGTGGAGTCGCAGACCAGAGCTGTCTGAACTCACGTGTCAGAAGCCTCCAAGCCTGCCT
 721 CCCAAGGTCCTCTCAGTTCTCTCCCTTCCTCTCTCCTTATAGACACTTGCTCCCAACCCATTCACTACAGGTGAAGGCTC
 801 TCACCCCCATCCCTGGGGGCCTTGGGTGAGTGGCCTGCTAAGGCTCCTCCTTGCCCAGACTACAGGGCTTAGGACTTGGT
 881 TTGTTATATCAGGGAAAAGGAGTAGGGAGTTCATCTGGAGGGTTCTAAGTGGGAGAAGGACTATCAACACCACTAGGAAT
 961 CCCAGAGGTGGGATCCTCCCTCATGGCTCTGGCACAGTGTAATCCAGGGGTGTAGATGGGGGAACTGTGAATACTTGAAC
1041 TCTGTTCCCCCACCCTCCATGCTCCTCACCTGTCTAGGTCTCCTCAGGGTGGGGGGTGACAGTGCCTTCTCTATTGGGCA
1121 CAGCCTAGGGTCTTGGGGGTCAGGGGGGAGAAGTTCTTGATTCAGCCAAATGCAGGGAGGGGAGGCAGATGGAGCCCATA
1201 GGCCACCCCCTATCCTCTGAGTGTTTGGAAATAAACTGTGCAATCCCCTCACCCTGAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ugUCCGUACCCUCCAGUCCACu 5' :||: | ||:||||||| | Target 5' taGGGTCTTGGGGGTCAGGGGg 3'	1126 - 1147	140.00	-23.80
2	miRNA 3' ugucCGUACCCUC---CAGUCC-ACu 5' ||:||||:| |||||| || Target 5' ccctGCGTGGGGGCCAGTCAGGCTGc 3'	142 - 167	138.00	-24.50
3	miRNA 3' ugUCCGUACCCUCCAGUCCACu 5' :||| ||||: || :|||| Target 5' ctGGGCTTGGGGTGTGGGGTGg 3'	640 - 661	128.00	-24.90

Mutant ID	Mutant Position	Mutant Source
COSN30473603	15	COSMIC
COSN31552334	26	COSMIC
COSN19728699	27	COSMIC
COSN30466588	38	COSMIC
COSN31511698	65	COSMIC
COSN28644632	73	COSMIC
COSN31586608	105	COSMIC
COSN30166831	113	COSMIC
COSN30125885	134	COSMIC
COSN7870976	138	COSMIC
COSN24301449	212	COSMIC
COSN7870975	233	COSMIC
COSN15118594	277	COSMIC
COSN31521542	366	COSMIC
COSN31568459	517	COSMIC
COSN505865	891	COSMIC
COSN31532947	1032	COSMIC
COSN30539097	1193	COSMIC
rs511515	73	GWAS
rs210135	888	GWAS

Mutant ID	Mutant Position	Mutant Source
rs1426714534	4	dbSNP
rs376265958	7	dbSNP
rs778565612	8	dbSNP
rs1472900723	10	dbSNP
rs755412880	11	dbSNP
rs754210197	16	dbSNP
rs767030876	26	dbSNP
rs7341238	27	dbSNP
rs1206414817	39	dbSNP
rs1342102424	41	dbSNP
rs1270919706	43	dbSNP
rs1230138313	46	dbSNP
rs181393571	47	dbSNP
rs531658995	53	dbSNP
rs936340521	54	dbSNP
rs1239818966	58	dbSNP
rs183363282	60	dbSNP
rs1424203495	65	dbSNP
rs190767037	72	dbSNP
rs511515	73	dbSNP
rs1030271610	85	dbSNP
rs1175373391	85	dbSNP
rs1207508382	88	dbSNP
rs992275709	89	dbSNP
rs8180574	92	dbSNP
rs553427506	93	dbSNP
rs1458714295	97	dbSNP
rs959839194	105	dbSNP
rs541193872	110	dbSNP
rs1343488901	116	dbSNP
rs577008922	125	dbSNP
rs1264548915	131	dbSNP
rs558742403	134	dbSNP
rs1221288736	135	dbSNP
rs2894353	147	dbSNP
rs537321467	148	dbSNP
rs570018194	150	dbSNP
rs1225924323	168	dbSNP
rs5745596	172	dbSNP
rs1315777484	180	dbSNP
rs2395456	185	dbSNP
rs982841564	186	dbSNP
rs1198279189	188	dbSNP
rs1252215085	193	dbSNP
rs1473479636	209	dbSNP
rs1175382797	216	dbSNP
rs955428407	219	dbSNP
rs1400183367	225	dbSNP
rs1029775599	229	dbSNP
rs536238538	230	dbSNP
rs1367531770	241	dbSNP
rs5745597	242	dbSNP
rs900366697	246	dbSNP
rs868651398	250	dbSNP
rs547448643	264	dbSNP
rs1338864089	277	dbSNP
rs1351462077	282	dbSNP
rs1017485229	286	dbSNP
rs1468404232	289	dbSNP
rs1358856165	291	dbSNP
rs36040266	291	dbSNP
rs1170822781	295	dbSNP
rs1001191226	307	dbSNP
rs903503254	308	dbSNP
rs1417219784	310	dbSNP
rs1042048544	312	dbSNP
rs1445224397	313	dbSNP
rs1244439239	316	dbSNP
rs1483913095	325	dbSNP
rs1202257617	333	dbSNP
rs1201270730	334	dbSNP
rs1482710486	336	dbSNP
rs945074930	337	dbSNP
rs529272226	341	dbSNP
rs2894354	344	dbSNP
rs1387111089	345	dbSNP
rs1205610981	360	dbSNP
rs1163136458	361	dbSNP
rs866811278	365	dbSNP
rs8180572	367	dbSNP
rs1371474218	371	dbSNP
rs1411987546	383	dbSNP
rs532258582	384	dbSNP
rs1327201626	387	dbSNP
rs1056657787	392	dbSNP
rs938187343	397	dbSNP
rs1339330533	407	dbSNP
rs571436982	408	dbSNP
rs1244694401	409	dbSNP
rs5745598	412	dbSNP
rs1307665059	418	dbSNP
rs1340971952	420	dbSNP
rs974313598	422	dbSNP
rs6457732	431	dbSNP
rs941626159	436	dbSNP
rs908821944	437	dbSNP
rs531593162	445	dbSNP
rs1241240641	450	dbSNP
rs1460033529	454	dbSNP
rs1183128182	456	dbSNP
rs1394389154	459	dbSNP
rs181851656	460	dbSNP
rs1298966574	463	dbSNP
rs955397475	465	dbSNP
rs1463278516	467	dbSNP
rs371998289	482	dbSNP
rs1029744520	492	dbSNP
rs1167800595	496	dbSNP
rs1425184374	512	dbSNP
rs975909018	513	dbSNP
rs1327708875	517	dbSNP
rs34299741	522	dbSNP
rs964536015	532	dbSNP
rs1269276740	539	dbSNP
rs1422755136	547	dbSNP
rs1225184668	550	dbSNP
rs1261163201	554	dbSNP
rs1322654477	555	dbSNP
rs1209281777	558	dbSNP
rs1395376781	559	dbSNP
rs1448864436	560	dbSNP
rs1191264858	561	dbSNP
rs1250725833	573	dbSNP
rs1241815781	576	dbSNP
rs1478554782	581	dbSNP
rs1178963618	583	dbSNP
rs1178567310	584	dbSNP
rs1017871840	586	dbSNP
rs1000760266	603	dbSNP
rs1160576180	604	dbSNP
rs1479494135	633	dbSNP
rs1386461449	651	dbSNP
rs1272897038	656	dbSNP
rs1317188402	658	dbSNP
rs903768325	659	dbSNP
rs549053288	660	dbSNP
rs1281065882	664	dbSNP
rs1344661805	670	dbSNP
rs1294625586	671	dbSNP
rs1009187479	680	dbSNP
rs530082443	681	dbSNP
rs1352932036	687	dbSNP
rs1203532110	694	dbSNP
rs7746159	695	dbSNP
rs896192354	696	dbSNP
rs1321209815	697	dbSNP
rs1201907318	700	dbSNP
rs1307870656	701	dbSNP
rs768421930	705	dbSNP
rs756795792	706	dbSNP
rs993882511	714	dbSNP
rs7762214	725	dbSNP
rs905336770	727	dbSNP
rs1038957476	733	dbSNP
rs1300961171	736	dbSNP
rs1364698387	738	dbSNP
rs1410275743	742	dbSNP
rs191850317	744	dbSNP
rs1333848430	766	dbSNP
rs908790799	773	dbSNP
rs1278990137	775	dbSNP
rs1047378690	783	dbSNP
rs933945489	785	dbSNP
rs1490288242	787	dbSNP
rs1196804787	788	dbSNP
rs1405350622	789	dbSNP
rs1253121538	791	dbSNP
rs568388557	804	dbSNP
rs975497130	805	dbSNP
rs1161595906	810	dbSNP
rs964487805	819	dbSNP
rs1445969126	822	dbSNP
rs1161276368	832	dbSNP
rs1384285814	844	dbSNP
rs7741608	845	dbSNP
rs1462189301	863	dbSNP
rs1457400673	864	dbSNP
rs1299600554	886	dbSNP
rs210135	888	dbSNP
rs1383579598	897	dbSNP
rs1248353333	900	dbSNP
rs1313175781	921	dbSNP
rs1160712491	925	dbSNP
rs1416920495	930	dbSNP
rs1250646259	936	dbSNP
rs1181864913	939	dbSNP
rs979658273	941	dbSNP
rs546993172	942	dbSNP
rs1490617457	953	dbSNP
rs1479876048	972	dbSNP
rs1195700040	975	dbSNP
rs967896055	980	dbSNP
rs1020821711	982	dbSNP
rs565090846	983	dbSNP
rs960324325	993	dbSNP
rs1356344850	996	dbSNP
rs1172114035	1009	dbSNP
rs1371103276	1012	dbSNP
rs543617808	1018	dbSNP
rs576272583	1022	dbSNP
rs1001933388	1027	dbSNP
rs1241333545	1028	dbSNP
rs1408287948	1032	dbSNP
rs1450376944	1040	dbSNP
rs1334706492	1049	dbSNP
rs1327274126	1050	dbSNP
rs1287995396	1051	dbSNP
rs1382942714	1055	dbSNP
rs1298051291	1060	dbSNP
rs1306120294	1064	dbSNP
rs528558662	1068	dbSNP
rs555242575	1070	dbSNP
rs1347149522	1091	dbSNP
rs1321423417	1092	dbSNP
rs564580110	1093	dbSNP
rs1485934557	1096	dbSNP
rs4713635	1100	dbSNP
rs1038161127	1102	dbSNP
rs1005715426	1103	dbSNP
rs867733152	1114	dbSNP
rs1047303005	1119	dbSNP
rs536574362	1141	dbSNP
rs1175645019	1143	dbSNP
rs546370243	1143	dbSNP
rs1431896832	1144	dbSNP
rs1051940	1150	dbSNP
rs1284280945	1154	dbSNP
rs1051945	1156	dbSNP
rs942741406	1160	dbSNP
rs926030238	1178	dbSNP
rs978851940	1184	dbSNP
rs1051958	1188	dbSNP
rs1485304165	1195	dbSNP
rs186506282	1199	dbSNP
rs1051964	1205	dbSNP
rs1270719135	1206	dbSNP
rs4711330	1208	dbSNP
rs150482729	1209	dbSNP
rs1198594422	1211	dbSNP
rs1408701161	1212	dbSNP
rs1463602704	1214	dbSNP
rs987995190	1216	dbSNP
rs1343539109	1224	dbSNP
rs960626444	1225	dbSNP
rs1034974143	1237	dbSNP
rs80065855	1240	dbSNP
rs969114479	1243	dbSNP
rs1017059187	1248	dbSNP
rs1374617387	1250	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.