>PABPN1|NM_004643|3'UTR
   1 AAAAAGTGTGTATTAGGAGGAGAGAGAGGAAAAAAAGAGGAAAGAAGGAAAAAAAAAAGAATTAAAAAAAAAAAAAAGAA
  81 AAACAGAAGATGACCTTGATGGAAAAAAAATATTTTTTAAAAAAAAGATATACTGTGGAAGGGGGGAGAATCCCATAACT
 161 AACTGCTGAGGAGGGACCTGCTTTGGGGAGTAGGGGAAGGCCCAGGGAGTGGGGCAGGGGGCTGCTTATTCACTCTGGGG
 241 ATTCGCCATGGACACGTCTCAACTGCGCAAGCTGCTGCCCATGTTTCCCTGCCCCACTTCACCCCCTTGGGGGCTGCTCA
 321 AGGGTAGGTGGGCGTGGGTGGTAGGAGGTTTTTTTTTTTACCCAGGGCTCTGGAAGGACACCAAACTGTTCTGCTTGTTA
 401 CCTTCCCTCCGTCTTCTCCTCGCCTTTCACAGTCCCCTCCTGCCTGCTCCTGTCCAGCCAGGTCTACCACCCACCCCACC
 481 CCTCTTCTCCGGCTCCCTGCCCCTCCAGATTGCCTGGTGATCTATTTTGTTTCCTTTTGTGTTTCTTTTTCTGTTTTGAG
 561 TGTCTTTCTTTGCAGGTTTCTGTAGCCGGAAGATCTCCGTTCCGCTCCCAGCGGCTCCAGTGTAAATTCCCCTTCCCCCT
 641 GGGGAAATGCACTACCTTGTTTTGGGGGGTTTAGGGGTGTTTTTGTTTTTCAGTTGTTTTGTTTTTTTGTTTTTTTTTTT
 721 TTTCCTTTGCCTTTTTTCCCTTTTATTTGGAGGGAATGGGAGGAAGTGGGAACAGGGAGGTGGGAGGTGGATTTTGTTTA
 801 TTTTTTTAGCTCATTTCCAGGGGTGGGAATTTTTTTTTAATATGTGTCATGAATAAAGTTGTTTTTGAAAATAAAAATTG
 881 TTTGGCCTTTTGGGTGTAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ucauguaguagAUAUGACAu 5' |||||||| Target 5' taaaaaaaagaTATACTGTg 3'	118 - 137	145.00	-8.70
2	miRNA 3' ucaugUAGUAGAUAUGACAu 5' :| |||||| :||| Target 5' gcctgGTGATCTATTTTGTt 3'	512 - 531	115.00	-8.70
3	miRNA 3' ucauguaGUAGAUAUGACAu 5' || | | ||||| Target 5' ggaaggaCA-CCAAACTGTt 3'	372 - 390	112.00	-5.10

Mutant ID	Mutant Position	Mutant Source
COSM1685706	6	COSMIC
COSM8209215	6	COSMIC
COSN15667009	38	COSMIC
COSN18738429	60	COSMIC
COSN28209498	64	COSMIC
COSN32061408	78	COSMIC
COSN1165935	79	COSMIC
COSN30613278	111	COSMIC
COSN30695539	111	COSMIC
COSN13578623	119	COSMIC
COSN30496551	120	COSMIC
COSN26574568	144	COSMIC
COSN31594631	147	COSMIC
COSN31547666	149	COSMIC
COSN31531048	165	COSMIC
COSN26575308	196	COSMIC
COSN1165936	284	COSMIC
COSN31533667	292	COSMIC
COSN1640427	316	COSMIC
COSN20111109	360	COSMIC
COSN22327992	422	COSMIC
COSN19363454	540	COSMIC
COSN31485828	555	COSMIC
COSN31609626	589	COSMIC
COSN20111113	723	COSMIC
COSN20111111	724	COSMIC
COSN31522434	738	COSMIC
COSN31482473	808	COSMIC
COSN30543888	842	COSMIC
COSN31546853	852	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs144841285	1	dbSNP
rs1402719809	6	dbSNP
rs1282689230	7	dbSNP
rs747100665	16	dbSNP
rs1168626892	20	dbSNP
rs754022244	20	dbSNP
rs768850285	23	dbSNP
rs1347453573	25	dbSNP
rs1221815445	27	dbSNP
rs762783207	28	dbSNP
rs1318911171	29	dbSNP
rs1196106320	30	dbSNP
rs1274871248	32	dbSNP
rs1435936150	36	dbSNP
rs776757269	38	dbSNP
rs577048276	39	dbSNP
rs766130036	39	dbSNP
rs748378670	42	dbSNP
rs769858590	45	dbSNP
rs774229260	47	dbSNP
rs759255838	48	dbSNP
rs1167790907	49	dbSNP
rs751517593	49	dbSNP
rs752252926	49	dbSNP
rs754929166	49	dbSNP
rs780929696	49	dbSNP
rs1472110999	50	dbSNP
rs1462200253	51	dbSNP
rs371252638	59	dbSNP
rs373625324	59	dbSNP
rs201110573	60	dbSNP
rs202153798	63	dbSNP
rs1273235905	64	dbSNP
rs1456900635	64	dbSNP
rs376203495	64	dbSNP
rs1159524550	65	dbSNP
rs1404641897	73	dbSNP
rs1224063962	75	dbSNP
rs1054460772	79	dbSNP
rs1329497188	81	dbSNP
rs1338575344	85	dbSNP
rs985279040	93	dbSNP
rs1395381336	95	dbSNP
rs926848552	107	dbSNP
rs1295351633	112	dbSNP
rs915112122	113	dbSNP
rs545879467	117	dbSNP
rs1441877511	119	dbSNP
rs200118006	119	dbSNP
rs182814853	120	dbSNP
rs1176438723	132	dbSNP
rs1375341630	142	dbSNP
rs1042314567	143	dbSNP
rs1011358370	145	dbSNP
rs1253148284	148	dbSNP
rs1211101944	149	dbSNP
rs923573734	172	dbSNP
rs78041837	176	dbSNP
rs1216260205	178	dbSNP
rs903020242	181	dbSNP
rs34164276	185	dbSNP
rs939048014	187	dbSNP
rs1306784222	189	dbSNP
rs1239773357	191	dbSNP
rs1282890453	191	dbSNP
rs1321454582	202	dbSNP
rs997316903	205	dbSNP
rs80204954	211	dbSNP
rs1437730287	213	dbSNP
rs386775604	213	dbSNP
rs894880223	215	dbSNP
rs1390675099	217	dbSNP
rs953250540	219	dbSNP
rs530033115	221	dbSNP
rs1289921351	223	dbSNP
rs1203121669	230	dbSNP
rs1422024623	236	dbSNP
rs1234951185	240	dbSNP
rs1470726786	243	dbSNP
rs1007462960	245	dbSNP
rs765008147	246	dbSNP
rs963708603	256	dbSNP
rs1430899452	257	dbSNP
rs1177934473	263	dbSNP
rs1194368896	266	dbSNP
rs759640107	267	dbSNP
rs546789070	268	dbSNP
rs1044538123	272	dbSNP
rs1257932128	272	dbSNP
rs972179183	279	dbSNP
rs919303853	282	dbSNP
rs1330639092	283	dbSNP
rs1296360525	288	dbSNP
rs1355994175	294	dbSNP
rs1339081017	296	dbSNP
rs560367991	297	dbSNP
rs1402494017	299	dbSNP
rs532389197	301	dbSNP
rs926776293	302	dbSNP
rs1410243961	303	dbSNP
rs1464215448	304	dbSNP
rs1302729663	305	dbSNP
rs1170771733	307	dbSNP
rs1054084	313	dbSNP
rs1260867340	314	dbSNP
rs1015862114	316	dbSNP
rs961654587	318	dbSNP
rs1257973096	321	dbSNP
rs1198997689	322	dbSNP
rs998179710	326	dbSNP
rs1343852665	334	dbSNP
rs1030105971	335	dbSNP
rs1316606826	340	dbSNP
rs1278066697	341	dbSNP
rs1415812377	343	dbSNP
rs1342673279	349	dbSNP
rs1400969671	349	dbSNP
rs35455483	349	dbSNP
rs397852863	349	dbSNP
rs398024557	349	dbSNP
rs149299976	350	dbSNP
rs954102336	351	dbSNP
rs985583132	360	dbSNP
rs1421430721	361	dbSNP
rs1275512304	364	dbSNP
rs1338891502	367	dbSNP
rs1473073244	372	dbSNP
rs1213269335	378	dbSNP
rs1275724222	385	dbSNP
rs915043434	388	dbSNP
rs1442063868	392	dbSNP
rs564060814	396	dbSNP
rs552245849	398	dbSNP
rs1267268613	401	dbSNP
rs1200623637	410	dbSNP
rs1257467414	411	dbSNP
rs150958673	411	dbSNP
rs749257506	412	dbSNP
rs1359076664	413	dbSNP
rs769676403	416	dbSNP
rs1377683083	420	dbSNP
rs537806866	422	dbSNP
rs939291606	423	dbSNP
rs1166049632	424	dbSNP
rs1448493166	428	dbSNP
rs1397562874	435	dbSNP
rs1421597357	436	dbSNP
rs1405949972	437	dbSNP
rs755065610	451	dbSNP
rs77613634	454	dbSNP
rs947002959	458	dbSNP
rs1472313369	461	dbSNP
rs947766256	464	dbSNP
rs1162665002	467	dbSNP
rs1363560730	469	dbSNP
rs1237719777	470	dbSNP
rs1222316209	473	dbSNP
rs1453850196	478	dbSNP
rs1288295724	479	dbSNP
rs547890319	481	dbSNP
rs1041476215	482	dbSNP
rs14947	489	dbSNP
rs568385003	491	dbSNP
rs1311888376	492	dbSNP
rs1241944110	498	dbSNP
rs997366735	504	dbSNP
rs191663420	509	dbSNP
rs554081795	512	dbSNP
rs1005738831	515	dbSNP
rs1007516060	521	dbSNP
rs1016557968	524	dbSNP
rs866598554	525	dbSNP
rs577016839	526	dbSNP
rs1245935405	530	dbSNP
rs539694546	530	dbSNP
rs897393406	533	dbSNP
rs556576341	534	dbSNP
rs1191872680	542	dbSNP
rs1479027073	551	dbSNP
rs796225168	557	dbSNP
rs1362112572	561	dbSNP
rs998879063	567	dbSNP
rs1488896368	588	dbSNP
rs752333454	589	dbSNP
rs1214819358	590	dbSNP
rs1351067701	594	dbSNP
rs1283057584	596	dbSNP
rs993879193	600	dbSNP
rs1267652620	604	dbSNP
rs1404315349	608	dbSNP
rs757993574	613	dbSNP
rs970810205	614	dbSNP
rs1397858559	618	dbSNP
rs1360172169	622	dbSNP
rs1177108893	634	dbSNP
rs1249392178	638	dbSNP
rs982149610	640	dbSNP
rs1423224649	641	dbSNP
rs1172064611	642	dbSNP
rs1182977192	650	dbSNP
rs1006951059	651	dbSNP
rs1191978754	656	dbSNP
rs926828792	658	dbSNP
rs959528978	659	dbSNP
rs1238376962	661	dbSNP
rs1164006854	664	dbSNP
rs867458356	666	dbSNP
rs1399222881	668	dbSNP
rs968146958	670	dbSNP
rs977937592	671	dbSNP
rs915443991	679	dbSNP
rs576470434	682	dbSNP
rs1279975209	693	dbSNP
rs1031284571	697	dbSNP
rs1216690859	697	dbSNP
rs1320330653	697	dbSNP
rs773621711	698	dbSNP
rs1450543621	701	dbSNP
rs1357575838	702	dbSNP
rs960653009	702	dbSNP
rs992485914	702	dbSNP
rs1344898560	705	dbSNP
rs947086091	706	dbSNP
rs1452528674	708	dbSNP
rs916211954	709	dbSNP
rs1202522042	710	dbSNP
rs1259403513	710	dbSNP
rs1457857994	710	dbSNP
rs1477766385	710	dbSNP
rs201653941	710	dbSNP
rs398024558	710	dbSNP
rs58264930	710	dbSNP
rs71119016	710	dbSNP
rs74574876	710	dbSNP
rs749193731	710	dbSNP
rs773166413	710	dbSNP
rs774179209	710	dbSNP
rs796121388	710	dbSNP
rs878927408	710	dbSNP
rs200356031	711	dbSNP
rs1385353446	714	dbSNP
rs947738683	715	dbSNP
rs984484698	716	dbSNP
rs1349467992	717	dbSNP
rs908949379	718	dbSNP
rs940466748	719	dbSNP
rs1037195027	720	dbSNP
rs1491258347	723	dbSNP
rs1422398609	724	dbSNP
rs1491131952	724	dbSNP
rs879250269	724	dbSNP
rs1464969454	725	dbSNP
rs1202886307	726	dbSNP
rs1241236811	729	dbSNP
rs1425281251	730	dbSNP
rs1187967161	731	dbSNP
rs1239537150	732	dbSNP
rs1439908465	732	dbSNP
rs1363048598	738	dbSNP
rs897354921	738	dbSNP
rs1468791725	739	dbSNP
rs1161721071	740	dbSNP
rs1044475807	746	dbSNP
rs1223535118	747	dbSNP
rs1319744078	757	dbSNP
rs542174392	767	dbSNP
rs934221690	769	dbSNP
rs1051359042	773	dbSNP
rs1379388304	775	dbSNP
rs924253321	781	dbSNP
rs1307179167	782	dbSNP
rs1368957346	801	dbSNP
rs747411810	801	dbSNP
rs1006875924	802	dbSNP
rs933084019	803	dbSNP
rs1365408245	811	dbSNP
rs777368091	813	dbSNP
rs1159626030	818	dbSNP
rs1419936775	820	dbSNP
rs1335269355	823	dbSNP
rs183808166	824	dbSNP
rs1417958792	829	dbSNP
rs1000055543	830	dbSNP
rs1192900347	830	dbSNP
rs903929029	830	dbSNP
rs1306733886	837	dbSNP
rs1267993285	838	dbSNP
rs1030791837	839	dbSNP
rs1216982283	844	dbSNP
rs960828831	862	dbSNP
rs907338904	868	dbSNP
rs1238568958	873	dbSNP
rs1312902138	875	dbSNP
rs771408031	878	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.