>HOXC4|NM_153633|3'UTR
   1 AACATAACTCACACCCCTGCCCCCACCCCATGCCCCCACCCTCCCCTCACACACAAATTGACTCTTATTTATAGAATTTA
  81 ATATATATATATATATATATATATATAGGTTCTTTTCTCTCTTCCTCTCACCTTGTCCCTTGTCAGTTCCAAACAGACAA
 161 AACAGATAAACAAACAAGCCCCCTGCCCTCCTCTCCCTCCCACTGTTAAGGACCCTTTTAAGCATGTGATGTTGTCTTAG
 241 CATGGTACCTGCTGGGTGTTTTTTTTTAAAAGGCCATTTTGGGGGGTTATTTATTTTTTAAGAAAAAAAGCTGCAAAAAT
 321 TATATATTGCAAGGTGTGATGGTCTGGCTTGGGTGAATTTCAGGGGAAATGAGGAAAAGAAAAAAGGAAAGAAATTTTAA
 401 AGCCAATTCTCATCCTTCTCCTCCTCCTCCTTCCCCCCCTCTTTCCTTAGGCCTTTTGCATTGAAAATGCACCAGGGGAG
 481 GTTAGTGAGGGGGAAGTCATTTTAAGGAGAACAAAGCTATGAAGTTCTTTTGTATTATTGTTGGGGGGGGGTGTGGGAGG
 561 AGAGGGGGCGAAGACAGCAGACAAAGCTAAATGCATCTGGAGAGCCTCTCAGAGCTGTTCAGTTTGAGGAGCCAAAAGAA
 641 AATCAAAATGAACTTTCAGTTCAGAGAGGCAGTCTATAGGTAGAATCTCTCCCCACCCCTATCGTGGTTATTGTGTTTTT
 721 GGACTGAATTTACTTGATTATTGTAAAACTTGCAATAAAGAATTTTAGTGTCGATGTGAAATGCCCCGTGATCAATAATA
 801 AACCAGTGGATGTGAATTAGTTTTAAAAAAAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cggacguaguacuuuACGGGAGc 5' ||||||| Target 5' aacaaacaagcccccTGCCCTCc 3'	169 - 191	140.00	-11.20
2	miRNA 3' cggAC-GUAGUACUUUACGGGagc 5' || |: ::|||||||||| Target 5' tagTGTCGATGTGAAATGCCCcgt 3'	766 - 789	132.00	-20.70
3	miRNA 3' cgGAC-GUAGUACUUUACGGGAGc 5' ||| | || |||||| | Target 5' ccCTGCCCCCACCCCATGCCCCCa 3'	15 - 38	117.00	-14.82

Mutant ID	Mutant Position	Mutant Source
COSN30513197	30	COSMIC
COSN31484873	269	COSMIC
COSN21474356	303	COSMIC
COSN26557083	303	COSMIC
COSN22963860	318	COSMIC
COSN31601069	349	COSMIC
COSN26642649	366	COSMIC
COSN31517169	397	COSMIC
COSN5717260	416	COSMIC
COSN20109657	440	COSMIC
COSN26638368	459	COSMIC
COSN31567471	507	COSMIC
COSN20109662	552	COSMIC
COSN31488506	561	COSMIC
COSN26478914	587	COSMIC
COSN31517460	704	COSMIC
COSN30172887	729	COSMIC
COSN21154539	770	COSMIC
COSN31489661	773	COSMIC
rs11554014	735	GWAS

Mutant ID	Mutant Position	Mutant Source
rs1199109156	5	dbSNP
rs925907252	6	dbSNP
rs955864955	11	dbSNP
rs778603565	13	dbSNP
rs747781602	15	dbSNP
rs1490955169	18	dbSNP
rs907588838	19	dbSNP
rs1391201989	20	dbSNP
rs771330513	20	dbSNP
rs776947223	21	dbSNP
rs759898104	22	dbSNP
rs770219186	24	dbSNP
rs1313659411	25	dbSNP
rs955200964	26	dbSNP
rs776001739	27	dbSNP
rs1250035186	29	dbSNP
rs1261860591	30	dbSNP
rs763013978	30	dbSNP
rs764256283	31	dbSNP
rs1166055452	32	dbSNP
rs1370275345	33	dbSNP
rs987040571	39	dbSNP
rs1162995309	43	dbSNP
rs751879896	46	dbSNP
rs762098070	47	dbSNP
rs1371477177	48	dbSNP
rs1411868498	50	dbSNP
rs1446066474	52	dbSNP
rs1238449893	63	dbSNP
rs1458798991	71	dbSNP
rs549378437	73	dbSNP
rs1191231455	77	dbSNP
rs1491269544	80	dbSNP
rs1198853523	81	dbSNP
rs1214242087	81	dbSNP
rs1225643860	81	dbSNP
rs1279415828	81	dbSNP
rs60894549	81	dbSNP
rs71070803	81	dbSNP
rs71444831	81	dbSNP
rs72272573	81	dbSNP
rs988499444	81	dbSNP
rs1433120895	83	dbSNP
rs922322899	89	dbSNP
rs962134549	91	dbSNP
rs933771820	95	dbSNP
rs1222696385	105	dbSNP
rs1362372758	109	dbSNP
rs1379870340	118	dbSNP
rs1464456605	120	dbSNP
rs113651547	138	dbSNP
rs1336839037	145	dbSNP
rs1470634758	145	dbSNP
rs750298433	148	dbSNP
rs1175199974	168	dbSNP
rs920285770	170	dbSNP
rs1467338170	172	dbSNP
rs138869843	174	dbSNP
rs1366924449	176	dbSNP
rs1422053956	180	dbSNP
rs556489134	181	dbSNP
rs1044937854	183	dbSNP
rs1238497123	186	dbSNP
rs1205652028	187	dbSNP
rs1459998739	197	dbSNP
rs927888234	198	dbSNP
rs939155110	205	dbSNP
rs571588877	206	dbSNP
rs1307928821	210	dbSNP
rs1324586099	211	dbSNP
rs1361195550	212	dbSNP
rs1315601337	215	dbSNP
rs1454135803	216	dbSNP
rs1037922915	226	dbSNP
rs899416598	227	dbSNP
rs1335716697	231	dbSNP
rs1462173979	242	dbSNP
rs993882179	243	dbSNP
rs1356700922	244	dbSNP
rs1047736067	246	dbSNP
rs1386434402	249	dbSNP
rs891874904	252	dbSNP
rs1442900108	254	dbSNP
rs1264368417	255	dbSNP
rs1179560872	257	dbSNP
rs199529607	259	dbSNP
rs1306651706	268	dbSNP
rs539258056	269	dbSNP
rs1043048998	274	dbSNP
rs1270150852	275	dbSNP
rs1477097655	278	dbSNP
rs1313720998	281	dbSNP
rs904493440	282	dbSNP
rs1375080493	285	dbSNP
rs1296051080	287	dbSNP
rs996207584	287	dbSNP
rs1029208017	293	dbSNP
rs954947967	297	dbSNP
rs1385091346	301	dbSNP
rs1009061342	303	dbSNP
rs1015126539	304	dbSNP
rs961809489	314	dbSNP
rs76297541	321	dbSNP
rs781328641	323	dbSNP
rs1295339075	330	dbSNP
rs1357212415	342	dbSNP
rs1290863934	364	dbSNP
rs1239176557	367	dbSNP
rs1358573180	374	dbSNP
rs878868809	380	dbSNP
rs748646192	388	dbSNP
rs1342083035	389	dbSNP
rs1222844564	392	dbSNP
rs1346600844	395	dbSNP
rs1305351833	407	dbSNP
rs1023044990	410	dbSNP
rs1365539134	416	dbSNP
rs1289910255	420	dbSNP
rs927878363	422	dbSNP
rs1362107440	424	dbSNP
rs572917439	428	dbSNP
rs1480880539	429	dbSNP
rs969858068	431	dbSNP
rs11362453	433	dbSNP
rs1489779120	433	dbSNP
rs67954156	433	dbSNP
rs999995461	433	dbSNP
rs368226822	434	dbSNP
rs796940867	436	dbSNP
rs1214381818	438	dbSNP
rs539902943	438	dbSNP
rs555412379	439	dbSNP
rs199756525	440	dbSNP
rs1284758383	442	dbSNP
rs561060764	451	dbSNP
rs573719745	462	dbSNP
rs1258387147	474	dbSNP
rs1296974851	480	dbSNP
rs772472613	485	dbSNP
rs1288380683	486	dbSNP
rs1470307385	503	dbSNP
rs985197261	508	dbSNP
rs1408615959	510	dbSNP
rs1174485570	517	dbSNP
rs1479356567	524	dbSNP
rs910981186	528	dbSNP
rs943476731	533	dbSNP
rs1193882403	541	dbSNP
rs973584970	541	dbSNP
rs11362454	543	dbSNP
rs1264666780	543	dbSNP
rs1430248502	543	dbSNP
rs376243618	543	dbSNP
rs796605810	543	dbSNP
rs920814244	545	dbSNP
rs929560643	546	dbSNP
rs1048472756	547	dbSNP
rs796708532	549	dbSNP
rs950128100	550	dbSNP
rs1469470003	551	dbSNP
rs200962735	553	dbSNP
rs201221665	554	dbSNP
rs1044425466	563	dbSNP
rs1168285060	563	dbSNP
rs890707876	570	dbSNP
rs544222846	571	dbSNP
rs999963084	572	dbSNP
rs1187821863	576	dbSNP
rs1413405841	580	dbSNP
rs1032822601	588	dbSNP
rs891654029	594	dbSNP
rs1444755377	600	dbSNP
rs1244444926	601	dbSNP
rs1181437202	607	dbSNP
rs1441504244	607	dbSNP
rs1250713300	615	dbSNP
rs1010138697	617	dbSNP
rs748403449	621	dbSNP
rs897967042	629	dbSNP
rs562798864	632	dbSNP
rs1361753150	633	dbSNP
rs995019246	635	dbSNP
rs1451918272	651	dbSNP
rs1336608982	653	dbSNP
rs1331075217	654	dbSNP
rs1392966614	655	dbSNP
rs1167043610	667	dbSNP
rs1394024300	669	dbSNP
rs45448092	671	dbSNP
rs545203102	674	dbSNP
rs1386955810	678	dbSNP
rs1018012509	688	dbSNP
rs1472156188	690	dbSNP
rs962465409	703	dbSNP
rs980403682	704	dbSNP
rs117223623	705	dbSNP
rs960598925	708	dbSNP
rs759418840	710	dbSNP
rs920754842	718	dbSNP
rs929503819	721	dbSNP
rs983713444	724	dbSNP
rs913837122	727	dbSNP
rs11554014	735	dbSNP
rs1227828239	736	dbSNP
rs1351462952	737	dbSNP
rs549165481	747	dbSNP
rs1352412262	750	dbSNP
rs759286609	753	dbSNP
rs1240233248	766	dbSNP
rs567421127	773	dbSNP
rs936076342	776	dbSNP
rs544567787	779	dbSNP
rs531627305	782	dbSNP
rs45625633	788	dbSNP
rs571624608	789	dbSNP
rs1237223624	796	dbSNP
rs1483704685	800	dbSNP
rs1183759967	803	dbSNP
rs1010491219	807	dbSNP
rs1200640119	818	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.