>NARS|NM_004539|3'UTR
   1 CCATTTTCTCCAGAAGCGTGGAGGAAAGATTATGAAAGGAACAGGCTCTTTAAAAAAGAAAACAAAAAGCCAGAATCTTC
  81 CTTTTTTTGTTTCATTGGGGTTTCTCTTTCTGTTTTTCTTTCTACTACCATAAAAACTATCTCAAATCACCTGAACATCA
 161 AGTGATATTAAGGTTGTCATCTTAAGAAAAAATATCCATTTTTTTCTTAAGTTCGGGAAACAAAGTTCGGGGAAAATACC
 241 TGGCATGAAACTGTAGTTAGGGATACATTTCAGCATTTTACTCACTTTATCCAAGTTATTCATTTTATTCAAGTTATATG
 321 TATGTATAATTCAACAATTTTAGATTATGGTGTAAGATACTCCAGTAACTTATCTTTCTGTCCTTTTAAGTGTACCTTGA
 401 ATTCTTTGATTTATTTTATTGCATCAATGAATTAAAACAAAAATCTTGGGGGAAGAAATTGGCAATATCGTATAAAAATC
 481 TGCTCATATTAGAACACAGTATAATTCAGCAGTAAACACTAGAATCAAATGAATAGCCTTTTGTATCAGTTATTAATCTT
 561 TTCTAACTCTGCTTAGCTGCTAATAATCCTGAGGCATAGAAATTGAAGAATTTGTAAAAATAGAATTGCCTTAAAGGATT
 641 TGAAGTAAGAACATAATTTTGGGGAGAGTTTTTTAGTGATTCACAGTATCCCTCTTAGCATTAATTTAAGGTAAAGAGGC
 721 AGATTGATTTTCCCTCTTTCCTGGTAATTCCTAAGTAATTAAGAATAAATAAGTTCCAAAAGAAATTGTAGCTGGAATCT
 801 TAATAACAATTGTGAGTGGCTGTTTGAGTTGCCCCCACCATGTCCTTAGATCTAATCTGTGCTACCTTATTAACTCACAG
 881 CAGGCTTACTGAATGGCTTCATTTCAGATTTAGTTGATTTCTCCACCAAATGCATGTCATGTATTCTCAATAGGCTGTAT
 961 TCCCAGCAGTCAATAAATGAACACCCGTAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ucgUACAUGGGUGUCAc 5' :|| |::|||||| Target 5' ttaGTG-ATTCACAGTa 3'	673 - 688	133.00	-10.30
2	miRNA 3' ucguACAU--GGGU-GUCAc 5' |||| |||| |||| Target 5' aggcTGTATTCCCAGCAGTc 3'	952 - 971	123.00	-12.50
3	miRNA 3' ucguacauggGUGUCAc 5' |||||| Target 5' catattagaaCACAGTa 3'	485 - 501	120.00	-6.40

Mutant ID	Mutant Position	Mutant Source
COSN18741442	18	COSMIC
COSN31558457	53	COSMIC
COSN28873126	82	COSMIC
COSN30153221	106	COSMIC
COSN21483735	113	COSMIC
COSN6128600	688	COSMIC
COSN8550615	946	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs778758664	2	dbSNP
rs754649223	3	dbSNP
rs1319147634	5	dbSNP
rs749697287	7	dbSNP
rs1214887805	11	dbSNP
rs200604529	12	dbSNP
rs374792914	17	dbSNP
rs2070109	18	dbSNP
rs1471577606	19	dbSNP
rs1266887754	20	dbSNP
rs1477725625	26	dbSNP
rs1270399196	34	dbSNP
rs1178032884	35	dbSNP
rs768003928	36	dbSNP
rs1487938330	37	dbSNP
rs367588725	39	dbSNP
rs898137656	41	dbSNP
rs751613291	44	dbSNP
rs367826619	45	dbSNP
rs1267903165	47	dbSNP
rs201761565	69	dbSNP
rs570743069	69	dbSNP
rs1304827503	70	dbSNP
rs146069417	71	dbSNP
rs1203984805	72	dbSNP
rs939927582	75	dbSNP
rs1279843016	76	dbSNP
rs1375088711	89	dbSNP
rs375966862	92	dbSNP
rs1237422509	95	dbSNP
rs917872065	105	dbSNP
rs1360553990	106	dbSNP
rs774698734	112	dbSNP
rs1288311147	123	dbSNP
rs1015387733	129	dbSNP
rs1225481343	138	dbSNP
rs746398673	139	dbSNP
rs774950385	144	dbSNP
rs980185746	156	dbSNP
rs770382291	157	dbSNP
rs534041561	162	dbSNP
rs572928154	168	dbSNP
rs1444997994	176	dbSNP
rs914560858	182	dbSNP
rs554879775	186	dbSNP
rs1404637833	187	dbSNP
rs1048601227	189	dbSNP
rs796696546	193	dbSNP
rs536120547	195	dbSNP
rs937783880	198	dbSNP
rs1444151324	206	dbSNP
rs1308043679	207	dbSNP
rs1339285401	208	dbSNP
rs904851771	214	dbSNP
rs1046104015	215	dbSNP
rs1019840504	228	dbSNP
rs748814352	229	dbSNP
rs1456465231	232	dbSNP
rs1211249498	233	dbSNP
rs867940754	234	dbSNP
rs949579682	235	dbSNP
rs1059593	239	dbSNP
rs914159944	243	dbSNP
rs1166556170	246	dbSNP
rs1461656004	252	dbSNP
rs1189346589	266	dbSNP
rs988299923	270	dbSNP
rs936823128	274	dbSNP
rs1028928496	281	dbSNP
rs1472443402	287	dbSNP
rs995249807	292	dbSNP
rs79936743	302	dbSNP
rs986174680	306	dbSNP
rs953495233	319	dbSNP
rs1036654141	324	dbSNP
rs1454194017	326	dbSNP
rs1030252388	327	dbSNP
rs976057350	331	dbSNP
rs1184129053	332	dbSNP
rs1228826668	335	dbSNP
rs1272413480	337	dbSNP
rs1003794825	344	dbSNP
rs755834460	347	dbSNP
rs747963459	350	dbSNP
rs372646072	364	dbSNP
rs1325931670	373	dbSNP
rs1056280609	380	dbSNP
rs1197363093	383	dbSNP
rs1253845035	384	dbSNP
rs937943187	386	dbSNP
rs1193857049	396	dbSNP
rs11356	398	dbSNP
rs1476586418	408	dbSNP
rs1169355003	413	dbSNP
rs538747287	414	dbSNP
rs1429301278	426	dbSNP
rs1043980527	427	dbSNP
rs571404920	437	dbSNP
rs1170399367	439	dbSNP
rs376424032	440	dbSNP
rs1402008917	441	dbSNP
rs142285081	444	dbSNP
rs1245264856	448	dbSNP
rs947484089	465	dbSNP
rs1317332314	469	dbSNP
rs528719755	470	dbSNP
rs147559695	471	dbSNP
rs183653451	472	dbSNP
rs1335734859	484	dbSNP
rs1231399979	485	dbSNP
rs1255595233	488	dbSNP
rs549672934	490	dbSNP
rs1344117651	494	dbSNP
rs1205486267	503	dbSNP
rs1287819267	509	dbSNP
rs3200342	518	dbSNP
rs535944092	519	dbSNP
rs1192804802	521	dbSNP
rs776107004	524	dbSNP
rs751413287	528	dbSNP
rs1196360038	533	dbSNP
rs904946359	539	dbSNP
rs1376712484	545	dbSNP
rs766432068	547	dbSNP
rs1160839738	549	dbSNP
rs1358978422	554	dbSNP
rs1419647046	563	dbSNP
rs955992604	571	dbSNP
rs1369867656	573	dbSNP
rs1385147984	579	dbSNP
rs373921197	582	dbSNP
rs1422440093	584	dbSNP
rs146942918	597	dbSNP
rs1311227086	599	dbSNP
rs987047691	601	dbSNP
rs1163725756	611	dbSNP
rs954466251	612	dbSNP
rs1013273442	613	dbSNP
rs772614070	614	dbSNP
rs892160895	621	dbSNP
rs757339017	623	dbSNP
rs936976463	629	dbSNP
rs925434380	635	dbSNP
rs58950020	638	dbSNP
rs187385712	640	dbSNP
rs1378905300	641	dbSNP
rs1222020114	645	dbSNP
rs1439439299	653	dbSNP
rs1489932371	661	dbSNP
rs1352105937	664	dbSNP
rs1456784739	668	dbSNP
rs1294013797	669	dbSNP
rs923375292	671	dbSNP
rs1449313967	674	dbSNP
rs1228983938	675	dbSNP
rs1307323848	675	dbSNP
rs976026204	684	dbSNP
rs369573428	686	dbSNP
rs1035120575	691	dbSNP
rs1350536045	719	dbSNP
rs1002111832	720	dbSNP
rs559827463	723	dbSNP
rs1480659736	741	dbSNP
rs907910330	748	dbSNP
rs983097961	754	dbSNP
rs952668155	755	dbSNP
rs180883037	756	dbSNP
rs1043566158	758	dbSNP
rs1457642001	771	dbSNP
rs946495535	772	dbSNP
rs1416321172	783	dbSNP
rs1422611171	788	dbSNP
rs1026781677	795	dbSNP
rs1164635942	796	dbSNP
rs1002058879	797	dbSNP
rs1241544790	799	dbSNP
rs1351487106	804	dbSNP
rs1373771130	806	dbSNP
rs969100272	810	dbSNP
rs1402698577	811	dbSNP
rs573007756	814	dbSNP
rs574211704	822	dbSNP
rs141508224	823	dbSNP
rs150135633	823	dbSNP
rs1329641977	826	dbSNP
rs1059645	837	dbSNP
rs1399818912	837	dbSNP
rs1453579471	840	dbSNP
rs1367079980	843	dbSNP
rs1303216442	851	dbSNP
rs1059648	852	dbSNP
rs1381022607	857	dbSNP
rs1394267437	870	dbSNP
rs1311878803	879	dbSNP
rs1318080234	880	dbSNP
rs1219921160	883	dbSNP
rs1053166847	890	dbSNP
rs1316924117	896	dbSNP
rs73961923	915	dbSNP
rs1059668	925	dbSNP
rs912550073	931	dbSNP
rs1013363190	933	dbSNP
rs1382621844	934	dbSNP
rs892120864	940	dbSNP
rs764397185	945	dbSNP
rs1001487841	949	dbSNP
rs1263044762	950	dbSNP
rs904113794	953	dbSNP
rs1050530045	956	dbSNP
rs536548758	957	dbSNP
rs1428300704	961	dbSNP
rs932164972	963	dbSNP
rs921475586	964	dbSNP
rs1360721640	982	dbSNP
rs575569414	984	dbSNP
rs1197070912	986	dbSNP
rs1479550362	987	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.