>REPIN1|NM_001099695|3'UTR
   1 GACGGTGGGCGGGGCCGTGTTGGCTGAGAGAGGGCTGGGGTCCTTCGTGGTGGGAGTCGCAGTGGGCTGGGGGTGCCTGC
  81 CTAGTGCTGGAGTAGGGGACAATGGGAATCCTAGAGGGGATGGAAGACGCGGGGAGTGAGCTGGGTGGGCCCTGCTAGCG
 161 AGAGAGGTCAACCCCGGTGGCCAGGGAACCCACTTCCAAGCGCAGGGACGCCGGCCTCCAGCTGGTGTGTGCTAAGGCTC
 241 CGTCCTGACTGCCCTGTGCCCTGGAAAAGCAGCAATAGCATCCGCCCCTTAGAGCCCTCTGGCTAGAGGAGCCACCAGTG
 321 GAAAGGAAGACCCTCCATCCTCTGGTATTAACGCCTTAATGCCCCTGTCTTTTACTGTAAGTTACTTAAGATCATTTTTG
 401 GAAGCAGGCGTGGTAGAGTCCTGTAAATGAATGCTCTGGGCTAGATACAGCTTGGAGAACCTGCTGGCCTTGTTAGACAG
 481 CACTTGGGCCTTTGCCAGCAGCAAGAGGTGAAGCGAAGCCACTCTTACCTCTCCCTTCCCCTCCCACCTGCCCCCTGCGT
 561 AGGCACCCAGACTTGGAGAGACCCGTCTGCTGTTAATACTTCCATCCTCTTCCTTCCCAAAGAGCAGATCCCAAGGCATT
 641 TACTCCTTGGTCTGTCTCGCTTTATCTGTCGCCCCTCCCAGCGCTGAGAGCCTCCCCTGGCTGTCAGCAGCACTGTGTCC
 721 AGGCTCTTGTCTGAACACCGCAGCCCCTCCTTCGCTCCTTCCAGAGCTCAGCATGTCACGGCAAGGACTGCCGCATTGGT
 801 GATGGAGGGCCAGCTGAGGGGAAGTTGCTGGTGAGTTTCCTTTTCTCCATTTCTAGCATATGGACACCTGGCCTCTGCTT
 881 GAGCACTTAGGTGACAGGAACTTCCGCACCTCCTGAGGCCCTGGATGATTCTAATTGTTAGAAATTCTAATTGTTAGAAA
 961 TCCTTCCTTATAATGAATGAATTCTGCTTTCCTATAATTTCTACCTATTGGGCCTTGTTCTGTTCTCTGGAACTAAACAG
1041 AACAACCATTTACCCCTCCTTTTCAAACTAGAGAATAAAGATTTGGTTTTAGAACTGGTAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' agcuGACGGUGAGAAUGAUCu 5' ||||:: |||::||| Target 5' gaacCTGCTGGCCTTGTTAGa 3'	457 - 477	129.00	-12.90
2	miRNA 3' agcuGACGGUGAGAAUGAUcu 5' |||: ||:|||||: Target 5' gcccCTGT--CTTTTACTGta 3'	361 - 379	121.00	-9.10
3	miRNA 3' agcUGACGG----UGAG---AAUGAUCu 5' |:||:: |:|| ||::||| Target 5' ctaATTGTTAGAAATTCTAATTGTTAGa 3'	931 - 958	116.00	-8.10

Mutant ID	Mutant Position	Mutant Source
COSN5853211	4	COSMIC
COSN30150870	5	COSMIC
COSN30478190	5	COSMIC
COSN31511209	11	COSMIC
COSN30501397	21	COSMIC
COSN30479150	27	COSMIC
COSN30518414	27	COSMIC
COSN19212826	59	COSMIC
COSN30108764	79	COSMIC
COSN15626115	111	COSMIC
COSN30510479	120	COSMIC
COSN31590436	130	COSMIC
COSN30133861	132	COSMIC
COSN31550540	145	COSMIC
COSN31778617	157	COSMIC
COSN31595313	181	COSMIC
COSN26556287	187	COSMIC
COSN31589541	202	COSMIC
COSN31542709	203	COSMIC
COSN22165177	241	COSMIC
COSN21701199	242	COSMIC
COSN25145949	264	COSMIC
COSN5651723	367	COSMIC
COSN30143484	421	COSMIC
COSN30529793	427	COSMIC
COSN30143121	490	COSMIC
COSN30510069	507	COSMIC
COSN21765485	552	COSMIC
COSN28741561	597	COSMIC
COSN28821792	630	COSMIC
COSN30531795	652	COSMIC
COSN30544093	772	COSMIC
COSN28821203	781	COSMIC
COSN31482096	783	COSMIC
COSN26552047	794	COSMIC
COSN30530678	820	COSMIC
COSN24302132	857	COSMIC
COSN30136282	962	COSMIC
COSN30112341	980	COSMIC
COSN9936614	1005	COSMIC
COSN31490790	1044	COSMIC
COSN31596646	1088	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1380067767	2	dbSNP
rs765174380	4	dbSNP
rs752590604	7	dbSNP
rs761921076	12	dbSNP
rs1281708597	15	dbSNP
rs767610161	18	dbSNP
rs1311996985	21	dbSNP
rs1239981165	26	dbSNP
rs1262753903	27	dbSNP
rs1484919365	31	dbSNP
rs1216054389	33	dbSNP
rs750666548	34	dbSNP
rs1420544708	37	dbSNP
rs1490474675	39	dbSNP
rs1201523129	42	dbSNP
rs1264946095	44	dbSNP
rs756351072	45	dbSNP
rs1023759899	50	dbSNP
rs1159340452	51	dbSNP
rs1181595883	54	dbSNP
rs1487192675	59	dbSNP
rs1471676696	62	dbSNP
rs1234512847	69	dbSNP
rs551774111	72	dbSNP
rs568786439	74	dbSNP
rs907499331	75	dbSNP
rs1212425220	102	dbSNP
rs116293076	103	dbSNP
rs554480290	111	dbSNP
rs73474368	112	dbSNP
rs534041526	118	dbSNP
rs1215108791	119	dbSNP
rs899106310	121	dbSNP
rs183206903	123	dbSNP
rs1226005755	124	dbSNP
rs961330211	126	dbSNP
rs1417727264	128	dbSNP
rs972941907	131	dbSNP
rs573297366	136	dbSNP
rs919762643	137	dbSNP
rs890324662	139	dbSNP
rs780101097	142	dbSNP
rs545392240	149	dbSNP
rs558970006	150	dbSNP
rs1324326298	151	dbSNP
rs1463176523	168	dbSNP
rs1395461565	170	dbSNP
rs1428171894	172	dbSNP
rs1165685602	174	dbSNP
rs575742388	182	dbSNP
rs1459912379	184	dbSNP
rs761453488	184	dbSNP
rs1008943576	185	dbSNP
rs910998536	186	dbSNP
rs1177643973	189	dbSNP
rs1325215966	190	dbSNP
rs1389270645	195	dbSNP
rs1477662385	196	dbSNP
rs1257083266	203	dbSNP
rs1031511932	204	dbSNP
rs1437117507	210	dbSNP
rs956703349	213	dbSNP
rs1195300257	218	dbSNP
rs1360965119	222	dbSNP
rs377426608	233	dbSNP
rs1314411084	250	dbSNP
rs1040729694	254	dbSNP
rs1332088728	259	dbSNP
rs561612183	262	dbSNP
rs969613960	265	dbSNP
rs530186400	267	dbSNP
rs1284364061	268	dbSNP
rs928116765	276	dbSNP
rs1054487905	284	dbSNP
rs1301940154	287	dbSNP
rs188141580	300	dbSNP
rs948233277	302	dbSNP
rs191964359	303	dbSNP
rs1320140998	305	dbSNP
rs907630653	306	dbSNP
rs1346977183	319	dbSNP
rs1159948686	320	dbSNP
rs1413794891	336	dbSNP
rs1393158974	337	dbSNP
rs879228245	346	dbSNP
rs1211078840	350	dbSNP
rs940345152	351	dbSNP
rs1485732666	356	dbSNP
rs1184346501	365	dbSNP
rs1182702009	366	dbSNP
rs1486656625	394	dbSNP
rs1242150813	401	dbSNP
rs1037312295	406	dbSNP
rs906632885	411	dbSNP
rs3192130	413	dbSNP
rs1237234305	417	dbSNP
rs1348962012	427	dbSNP
rs931895699	446	dbSNP
rs551884072	447	dbSNP
rs1164320716	448	dbSNP
rs1364383883	450	dbSNP
rs1014932531	451	dbSNP
rs1289709731	473	dbSNP
rs1447359580	478	dbSNP
rs768516226	482	dbSNP
rs1458784116	483	dbSNP
rs1314639625	490	dbSNP
rs897021870	503	dbSNP
rs1156782072	506	dbSNP
rs1344442902	515	dbSNP
rs994095840	516	dbSNP
rs774189227	526	dbSNP
rs1168096957	527	dbSNP
rs1448446831	529	dbSNP
rs1304300766	530	dbSNP
rs1353189819	533	dbSNP
rs375821873	533	dbSNP
rs890260300	536	dbSNP
rs1009160412	541	dbSNP
rs1052503991	545	dbSNP
rs1486296627	552	dbSNP
rs568498431	553	dbSNP
rs1288459617	560	dbSNP
rs1026891602	561	dbSNP
rs952533991	562	dbSNP
rs1309682645	567	dbSNP
rs1296544685	574	dbSNP
rs892525096	575	dbSNP
rs761570199	583	dbSNP
rs1353568209	586	dbSNP
rs1330542541	588	dbSNP
rs1051760	597	dbSNP
rs1051764	630	dbSNP
rs1316765242	632	dbSNP
rs1369841624	639	dbSNP
rs969418059	650	dbSNP
rs1463322931	655	dbSNP
rs965167043	656	dbSNP
rs184568102	665	dbSNP
rs960879533	669	dbSNP
rs1419323594	670	dbSNP
rs760702502	671	dbSNP
rs1471999755	672	dbSNP
rs1250404223	680	dbSNP
rs1481821246	681	dbSNP
rs983373547	684	dbSNP
rs923628099	691	dbSNP
rs908734597	703	dbSNP
rs935027674	708	dbSNP
rs1333005743	715	dbSNP
rs989737453	723	dbSNP
rs1233954753	729	dbSNP
rs973162033	731	dbSNP
rs1350017567	735	dbSNP
rs533709234	740	dbSNP
rs915521351	742	dbSNP
rs553953335	746	dbSNP
rs948178014	754	dbSNP
rs1473594403	755	dbSNP
rs1045270143	756	dbSNP
rs1396178816	757	dbSNP
rs985981669	765	dbSNP
rs911816497	769	dbSNP
rs906580272	774	dbSNP
rs1361628207	780	dbSNP
rs1051823	781	dbSNP
rs568607326	794	dbSNP
rs1251640199	796	dbSNP
rs543350624	806	dbSNP
rs1467455888	808	dbSNP
rs1255110917	810	dbSNP
rs1177986685	811	dbSNP
rs995275225	820	dbSNP
rs753150848	821	dbSNP
rs758877529	822	dbSNP
rs946779412	840	dbSNP
rs1302629599	845	dbSNP
rs1438410413	848	dbSNP
rs1372984773	853	dbSNP
rs1043837892	859	dbSNP
rs753749104	861	dbSNP
rs1433911174	865	dbSNP
rs752672882	877	dbSNP
rs1351854493	890	dbSNP
rs536005303	895	dbSNP
rs1159828677	904	dbSNP
rs559132783	905	dbSNP
rs1364859501	906	dbSNP
rs1018066578	919	dbSNP
rs1173626828	927	dbSNP
rs1278363823	928	dbSNP
rs371626146	928	dbSNP
rs965628555	933	dbSNP
rs575704271	936	dbSNP
rs1446664256	937	dbSNP
rs760061449	950	dbSNP
rs1259691673	964	dbSNP
rs1206936054	968	dbSNP
rs976523529	971	dbSNP
rs1373253305	972	dbSNP
rs1220260184	984	dbSNP
rs1349696006	987	dbSNP
rs765732778	993	dbSNP
rs189325156	998	dbSNP
rs1324290013	1001	dbSNP
rs1319936753	1003	dbSNP
rs1346117575	1005	dbSNP
rs956382460	1007	dbSNP
rs1004498391	1009	dbSNP
rs14356	1014	dbSNP
rs1275590409	1015	dbSNP
rs11764897	1029	dbSNP
rs575090642	1035	dbSNP
rs540446106	1041	dbSNP
rs192583157	1049	dbSNP
rs115575718	1052	dbSNP
rs1377605927	1054	dbSNP
rs1232321610	1059	dbSNP
rs1174686578	1068	dbSNP
rs1475312693	1071	dbSNP
rs948291741	1082	dbSNP
rs980894583	1083	dbSNP
rs928148818	1087	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.