>MSC|NM_005098|3'UTR
   1 ATCGGACTGGAACTCACTTGATGGGATTATTCGTTAAATGCGAGTGTTTGGGGGCCACGGAGAGAAGGGAGAGCTCGTGA
  81 GATGGGAAGAAGTTTCCGCTGGATTCTCCTTGACCCTTCCCCTTTCCCTGGAACTGTGATCGTGACAGGTGGCGGGTGTG
 161 GCTGTCACTGCACAGCGCCCACGGCTACAGCTGCGCCGGATCTGGGCGACCACGTTTTGCCTCTCCAAAAAGAGCTTCCT
 241 TTCGTGACGAGACGCGGACGCAGGTCCACCCTCGGGCCCTAGCTCTGTAGACTAACTCTCGGCTGCTGCCCCAGCCCGCG
 321 CCAGACAGCCCACGGATCCGTTCTCAGCGGAGCCAGATTCATCGCACACGTGCGGGACGGTTCCACACAGCCCCGGCCTT
 401 TCGCGGTGACACAATGGTTAGGGAACGGTTAGAACGCGCTCTACATCCGCTTTAAAGACAGAGGTCTAGACGTGAGATCC
 481 GCGTCGGGACAGGGTTTTAAGTGACAAAGAAGGGCGAGTGGCTTCTCTGGGCCGGGTTCGTACTCCAGCACAGCGCCCTT
 561 CTAACGGGCGGGAGGAAGGCCGCTGCTCGCAGGGCTAGGTGGAGACACACTTCCCAGATCACCGCAGGCGGGTTTTACCC
 641 GGAGAGCTCTGGGCCGTTCGGCCTCCCTGCCGGGTGGCTTCTTCAATCCCGTCTCCTTCCCAAGCTCCCGGCTTTTTCTA
 721 ATCAGGCAGGCGTCTGTCAACCCTCTCCACTTCTGGGCTGAAGCCTCCCCAAGCCCCGCTGCGCCAACCTGTGTGGGGTC
 801 TTCTTCGGGCCTCCCTCTCCGCCCCGCTCCTGCTCCTACCTGCAGCACCCCCAGCTCCGACTCCAGACTCTCTGCATCAG
 881 GTCTCCCCACTCCACGCTCCGGGCGCCCCAACTCCAACACCACGTCCTGCCGCGCAGGTTCTTCCCCGCGCGGAGGAGCG
 961 CGCAGGGTGGGCGGCTTACCATAGCAAGTGATCCTGCGATAGGGAACGCGCCCTTGCCCCGAGGCTGCACTACCACAGGA
1041 AATAACATATGTAAATAAATTTATTTTTTTATGAATAATAAAACGCGCTGTAAAAACCGTGTGCCCCTTGGAGGTGTCAA
1121 AAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' aggacUGGGGAU--CUGGGUUGAc 5' :|:|| : | ||||||| Target 5' tccacGCTCCGGGCGCCCCAACTc 3'	891 - 914	147.00	-17.40
2	miRNA 3' agGAC----UGGGGAUCUGGGUUGAc 5' ||| ||||| || :||:||| Target 5' acCTGCAGCACCCCCAGCTCCGACTc 3'	838 - 863	138.00	-24.70
3	miRNA 3' aggacUGGG-GAUCUGGGUUGAc 5' |||| | :| ||:|:|| Target 5' ggtccACCCTCGGGCCCTAGCTc 3'	263 - 285	125.00	-20.80

Mutant ID	Mutant Position	Mutant Source
COSN30153716	3	COSMIC
COSN26985131	4	COSMIC
COSN30666105	5	COSMIC
COSN4894180	20	COSMIC
COSN31494114	21	COSMIC
COSN30181214	22	COSMIC
COSN31501474	25	COSMIC
COSN31507945	26	COSMIC
COSN31527516	32	COSMIC
COSN509522	32	COSMIC
COSN30131130	33	COSMIC
COSN14352764	41	COSMIC
COSN26985133	51	COSMIC
COSN30106645	52	COSMIC
COSN30492268	53	COSMIC
COSN30498291	58	COSMIC
COSN30499907	59	COSMIC
COSN30139259	67	COSMIC
COSN26985132	68	COSMIC
COSN30485327	72	COSMIC
COSN30506514	76	COSMIC
COSN20076343	77	COSMIC
COSN31494785	84	COSMIC
COSN30501463	88	COSMIC
COSN30496546	91	COSMIC
COSN30178845	97	COSMIC
COSN26985134	98	COSMIC
COSN28726440	102	COSMIC
COSN30188185	109	COSMIC
COSN30456973	120	COSMIC
COSN30513267	122	COSMIC
COSN30155922	131	COSMIC
COSN30111092	153	COSMIC
COSN30191115	154	COSMIC
COSN30514797	161	COSMIC
COSN31613297	162	COSMIC
COSN30496105	177	COSMIC
COSN1367894	178	COSMIC
COSN30117450	193	COSMIC
COSN30188840	198	COSMIC
COSN30188866	199	COSMIC
COSN31498597	205	COSMIC
COSN27294057	207	COSMIC
COSN31510984	208	COSMIC
COSN30157729	223	COSMIC
COSN31554782	243	COSMIC
COSN23832769	244	COSMIC
COSN30506780	248	COSMIC
COSN31581624	253	COSMIC
COSN16551870	378	COSMIC
COSN17889829	426	COSMIC
COSN20150114	540	COSMIC
COSN8099437	553	COSMIC
COSN32207719	588	COSMIC
COSN16441309	623	COSMIC
COSN16745734	674	COSMIC
COSN2288096	678	COSMIC
COSN7052440	737	COSMIC
COSN23028505	783	COSMIC
COSN22197124	899	COSMIC
COSN15719514	914	COSMIC
COSN25019599	936	COSMIC
COSN6361888	977	COSMIC
COSN28198208	1064	COSMIC
rs3779760	257	GWAS

Mutant ID	Mutant Position	Mutant Source
rs764192144	3	dbSNP
rs756312389	15	dbSNP
rs752865426	16	dbSNP
rs765725019	16	dbSNP
rs1035939144	17	dbSNP
rs200639659	23	dbSNP
rs1412938768	26	dbSNP
rs1331191420	31	dbSNP
rs762714470	33	dbSNP
rs1355449592	40	dbSNP
rs750348703	40	dbSNP
rs560737829	41	dbSNP
rs1295190134	42	dbSNP
rs761869550	43	dbSNP
rs1021682607	50	dbSNP
rs935522389	53	dbSNP
rs540547503	56	dbSNP
rs1201081632	74	dbSNP
rs578226654	84	dbSNP
rs1011262026	89	dbSNP
rs190061014	94	dbSNP
rs1437001846	97	dbSNP
rs1202692485	98	dbSNP
rs982775538	99	dbSNP
rs899562763	106	dbSNP
rs1490481761	123	dbSNP
rs1270295122	127	dbSNP
rs1232026360	135	dbSNP
rs1377121550	136	dbSNP
rs1039458282	141	dbSNP
rs3779761	148	dbSNP
rs1023067005	152	dbSNP
rs372069924	156	dbSNP
rs575691530	165	dbSNP
rs1167051662	169	dbSNP
rs1367870395	170	dbSNP
rs1406065876	171	dbSNP
rs1303121910	172	dbSNP
rs890999805	173	dbSNP
rs759976877	176	dbSNP
rs1046886362	177	dbSNP
rs1225764317	178	dbSNP
rs1293612109	180	dbSNP
rs1027401023	181	dbSNP
rs993256237	182	dbSNP
rs1449929672	183	dbSNP
rs919872063	184	dbSNP
rs1038089290	199	dbSNP
rs897665471	207	dbSNP
rs1166998575	214	dbSNP
rs148153224	220	dbSNP
rs535819859	221	dbSNP
rs1389133144	223	dbSNP
rs889066252	228	dbSNP
rs1318361541	231	dbSNP
rs566902481	234	dbSNP
rs1411601447	241	dbSNP
rs1316767687	243	dbSNP
rs752120642	249	dbSNP
rs796101677	253	dbSNP
rs3779760	257	dbSNP
rs763352558	259	dbSNP
rs1352699099	269	dbSNP
rs1205666150	271	dbSNP
rs1285016032	272	dbSNP
rs1464702491	274	dbSNP
rs990209942	275	dbSNP
rs11552826	277	dbSNP
rs1245262780	277	dbSNP
rs963715673	282	dbSNP
rs1052531995	283	dbSNP
rs533834455	286	dbSNP
rs3779759	290	dbSNP
rs1159704492	292	dbSNP
rs1046616786	301	dbSNP
rs185602446	306	dbSNP
rs916863177	309	dbSNP
rs991560434	311	dbSNP
rs531540800	313	dbSNP
rs951749258	314	dbSNP
rs1451496336	318	dbSNP
rs1310247694	320	dbSNP
rs920305556	328	dbSNP
rs562936721	332	dbSNP
rs994038927	344	dbSNP
rs1239636894	345	dbSNP
rs1438933495	346	dbSNP
rs898393777	364	dbSNP
rs971805828	367	dbSNP
rs1206838862	368	dbSNP
rs1038321993	374	dbSNP
rs942021571	380	dbSNP
rs1199437803	385	dbSNP
rs549520951	388	dbSNP
rs1019180424	391	dbSNP
rs1421293352	392	dbSNP
rs3824153	394	dbSNP
rs1411954998	396	dbSNP
rs112401915	398	dbSNP
rs1234574799	404	dbSNP
rs1177913338	406	dbSNP
rs1352634176	416	dbSNP
rs1429403490	421	dbSNP
rs1306833098	422	dbSNP
rs1300755995	423	dbSNP
rs878918193	426	dbSNP
rs1398359650	427	dbSNP
rs1391813298	444	dbSNP
rs1444241690	448	dbSNP
rs938721703	456	dbSNP
rs953413212	463	dbSNP
rs928724911	464	dbSNP
rs776511442	468	dbSNP
rs1336314918	471	dbSNP
rs1333581398	472	dbSNP
rs1232768111	476	dbSNP
rs946264931	479	dbSNP
rs1196694578	481	dbSNP
rs1394439094	493	dbSNP
rs1377360723	509	dbSNP
rs1174437403	515	dbSNP
rs1010879957	518	dbSNP
rs1268938163	536	dbSNP
rs990456862	541	dbSNP
rs1378813728	543	dbSNP
rs900126	554	dbSNP
rs1462182867	555	dbSNP
rs1017865675	556	dbSNP
rs1368497967	557	dbSNP
rs753399580	561	dbSNP
rs1303731971	566	dbSNP
rs986446477	572	dbSNP
rs143686543	576	dbSNP
rs1299302865	577	dbSNP
rs906648229	581	dbSNP
rs139693900	584	dbSNP
rs1274333755	585	dbSNP
rs1186218898	589	dbSNP
rs1179570929	591	dbSNP
rs1225358854	595	dbSNP
rs578149674	600	dbSNP
rs994396804	605	dbSNP
rs1188879861	606	dbSNP
rs1263260433	614	dbSNP
rs1428693784	620	dbSNP
rs912897470	623	dbSNP
rs1440129667	624	dbSNP
rs1259019786	625	dbSNP
rs16937841	626	dbSNP
rs1426389230	627	dbSNP
rs948285865	631	dbSNP
rs148958311	638	dbSNP
rs370045145	640	dbSNP
rs1002898802	642	dbSNP
rs907273003	644	dbSNP
rs1041842720	646	dbSNP
rs575457599	648	dbSNP
rs555571882	649	dbSNP
rs1248546675	656	dbSNP
rs1291039660	665	dbSNP
rs1321600155	667	dbSNP
rs535575741	671	dbSNP
rs1485932208	673	dbSNP
rs181486064	677	dbSNP
rs1283462787	683	dbSNP
rs145691505	687	dbSNP
rs962080456	695	dbSNP
rs911682906	697	dbSNP
rs1412844919	704	dbSNP
rs1421959582	705	dbSNP
rs1313594236	706	dbSNP
rs987701512	709	dbSNP
rs911496467	723	dbSNP
rs573443891	731	dbSNP
rs1319985466	737	dbSNP
rs986415050	753	dbSNP
rs1454810265	757	dbSNP
rs955073443	758	dbSNP
rs1378964502	762	dbSNP
rs368733711	763	dbSNP
rs1029288013	764	dbSNP
rs1010848706	767	dbSNP
rs972469619	771	dbSNP
rs1265706665	776	dbSNP
rs1462350453	777	dbSNP
rs1203933901	778	dbSNP
rs777654941	787	dbSNP
rs533783989	789	dbSNP
rs1016740155	793	dbSNP
rs756146172	794	dbSNP
rs571407938	798	dbSNP
rs551667667	802	dbSNP
rs959258487	806	dbSNP
rs1472225567	807	dbSNP
rs999550737	813	dbSNP
rs1409877251	815	dbSNP
rs906607937	821	dbSNP
rs1471065739	823	dbSNP
rs755653797	824	dbSNP
rs538010476	825	dbSNP
rs1012795768	828	dbSNP
rs1358942732	829	dbSNP
rs895333544	830	dbSNP
rs1476485566	838	dbSNP
rs1300281790	839	dbSNP
rs1002869355	842	dbSNP
rs1228357066	844	dbSNP
rs1419334815	848	dbSNP
rs907242950	849	dbSNP
rs1487743366	853	dbSNP
rs1219439691	860	dbSNP
rs750366678	861	dbSNP
rs113340792	863	dbSNP
rs1310085866	868	dbSNP
rs368478178	874	dbSNP
rs931551957	877	dbSNP
rs1312660640	883	dbSNP
rs893227110	887	dbSNP
rs1054609113	895	dbSNP
rs1179986797	899	dbSNP
rs942898813	900	dbSNP
rs911463990	901	dbSNP
rs897435187	904	dbSNP
rs761667982	905	dbSNP
rs1424656533	906	dbSNP
rs1412987484	907	dbSNP
rs1172940977	931	dbSNP
rs940400828	934	dbSNP
rs1207948496	942	dbSNP
rs911595610	945	dbSNP
rs1280651603	947	dbSNP
rs558683533	948	dbSNP
rs931785571	958	dbSNP
rs921777755	959	dbSNP
rs1293962238	962	dbSNP
rs1218159161	963	dbSNP
rs1202932728	965	dbSNP
rs989344368	966	dbSNP
rs1293936880	971	dbSNP
rs972821766	978	dbSNP
rs549232169	979	dbSNP
rs957922754	980	dbSNP
rs1030962359	981	dbSNP
rs1272078102	984	dbSNP
rs751787869	990	dbSNP
rs1431768384	996	dbSNP
rs1246234444	999	dbSNP
rs1349612237	1007	dbSNP
rs1322019379	1015	dbSNP
rs1188560771	1021	dbSNP
rs1463442633	1023	dbSNP
rs529403794	1033	dbSNP
rs962469149	1034	dbSNP
rs978080884	1039	dbSNP
rs1167111066	1046	dbSNP
rs1367930501	1047	dbSNP
rs752030813	1050	dbSNP
rs1395912150	1051	dbSNP
rs1328898288	1052	dbSNP
rs1164987733	1058	dbSNP
rs1297963161	1064	dbSNP
rs1477172910	1064	dbSNP
rs909648313	1065	dbSNP
rs1316900303	1067	dbSNP
rs1416791873	1070	dbSNP
rs1209196226	1071	dbSNP
rs1025455894	1077	dbSNP
rs1289198686	1078	dbSNP
rs1012344644	1084	dbSNP
rs1210822283	1085	dbSNP
rs10093815	1086	dbSNP
rs1248811090	1086	dbSNP
rs1187860989	1087	dbSNP
rs1003423803	1098	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.