>GJB1|NM_000166|3'UTR
   1 TGCCACATACCAGGCAACCTCCCATCCCACCCCCGACCCTGCCCTGGGCGAGCCCCTCCTTCTCCCCTGCCGGTGCACAG
  81 GCCTCTGCCTGCTGGGGATTACTCGATCAAAACCTTCCTTCCCTGGCTACTTCCCTTCCTCCCGGGGCCTTCCTTTTGAG
 161 GAGCTGGAGGGGTGGGGAGCTAGAGGCCACCTATGCCAGTGCTCAAGGTTACTGGGAGTGTGGGCTGCCCTTGTTGCCTG
 241 CACCCTTCCCTCTTCCCTCTCCCTCTCTCTGGGACCACTGGGTACAAGAGATGGGATGCTCCGACAGCGTCTCCAATTAT
 321 GAAACTAATCTTAACCCTGTGCTGTCAGATACCCTGTTTCTGGAGTCACATCAGTGAGGAGGGATGTGGGTAAGAGGAGC
 401 AGAGGGCAGGGGTGCTGTGGACATGTGGGTGGAGAAGGGAGGGTGGCCAGCACTAGTAAAGGAGGAATAGTGCTTGCTGG
 481 CCACAAGGAAAAGGAGGAGGTGTCTGGGGTGAGGGAGTTAGGGAGAGAGAAGCAGGCAGATAAGTTGGAGCAGGGGTTGG
 561 TCAAGGCCACCTCTGCCTCTAGTCCCCAAGGCCTCTCTCTGCCTGAAATGTTACACATTAAACAGGATTTTACAGTAAAT
 641 GAAGAGGTGGCTTGTGAAAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' aacaGGUGAAG--AAGGACGGGAc 5' |||| | |||||||| Target 5' catcCCACCCCCGACCCTGCCCTg 3'	23 - 46	148.00	-22.30
2	miRNA 3' aacaggugaagaagGACGGGAc 5' ||||||| Target 5' actgggagtgtgggCTGCCCTt 3'	211 - 232	140.00	-11.50
3	miRNA 3' aacAGGUGAAGAAGGACGGGAc 5' || | |||||| |||| Target 5' cgaTCAAAACCTTCCTTCCCTg 3'	104 - 125	131.00	-19.40

Mutant ID	Mutant Position	Mutant Source
379903	16	ClinVar
917323	19	ClinVar
913989	23	ClinVar
368627	35	ClinVar
913990	51	ClinVar
368628	118	ClinVar
368629	164	ClinVar
915218	278	ClinVar
915219	310	ClinVar
915220	403	ClinVar
915221	444	ClinVar

Mutant ID	Mutant Position	Mutant Source
rs1382950579	5	dbSNP
rs1055605737	8	dbSNP
rs1057520778	16	dbSNP
rs769121326	20	dbSNP
rs745547989	23	dbSNP
rs1450931734	25	dbSNP
rs1314722080	27	dbSNP
rs1360833066	28	dbSNP
rs769526542	30	dbSNP
rs1042771	31	dbSNP
rs1282246691	33	dbSNP
rs1259454589	34	dbSNP
rs113014279	35	dbSNP
rs1372482578	35	dbSNP
rs762776007	36	dbSNP
rs1032823915	37	dbSNP
rs763949039	40	dbSNP
rs774210122	42	dbSNP
rs770941057	44	dbSNP
rs1181553807	45	dbSNP
rs1242256515	50	dbSNP
rs771578720	59	dbSNP
rs1311893378	69	dbSNP
rs774283188	73	dbSNP
rs1374004037	105	dbSNP
rs1369792450	106	dbSNP
rs41307256	118	dbSNP
rs185840896	119	dbSNP
rs897752511	120	dbSNP
rs1314584361	123	dbSNP
rs964774958	126	dbSNP
rs997612029	128	dbSNP
rs1252712791	144	dbSNP
rs1187450602	145	dbSNP
rs1379809909	146	dbSNP
rs760199887	147	dbSNP
rs139775308	164	dbSNP
rs559651431	183	dbSNP
rs1308871981	196	dbSNP
rs1228736552	214	dbSNP
rs764034005	219	dbSNP
rs1225463519	224	dbSNP
rs190185368	243	dbSNP
rs770512935	251	dbSNP
rs1341973897	257	dbSNP
rs1412410286	258	dbSNP
rs776265884	266	dbSNP
rs759936874	278	dbSNP
rs1239659935	303	dbSNP
rs757677225	304	dbSNP
rs181881705	310	dbSNP
rs1316836115	317	dbSNP
rs968982704	318	dbSNP
rs979721736	323	dbSNP
rs1164237138	346	dbSNP
rs1474271801	349	dbSNP
rs893533943	351	dbSNP
rs1011044023	353	dbSNP
rs1021045505	354	dbSNP
rs14974	355	dbSNP
rs938410367	369	dbSNP
rs1485421059	375	dbSNP
rs1181327701	376	dbSNP
rs966935943	379	dbSNP
rs1210492082	383	dbSNP
rs1451550993	401	dbSNP
rs753223616	403	dbSNP
rs187003364	410	dbSNP
rs1389797471	413	dbSNP
rs1035055668	424	dbSNP
rs1323011740	428	dbSNP
rs533182215	444	dbSNP
rs1178001997	446	dbSNP
rs1048261406	468	dbSNP
rs959616348	471	dbSNP
rs770160056	474	dbSNP
rs1361657825	478	dbSNP
rs1268834259	484	dbSNP
rs888344835	487	dbSNP
rs1389939284	497	dbSNP
rs1359625100	541	dbSNP
rs1351758937	546	dbSNP
rs1165715387	553	dbSNP
rs1428194429	556	dbSNP
rs1417665342	561	dbSNP
rs1449851024	572	dbSNP
rs990812243	577	dbSNP
rs1006838208	585	dbSNP
rs758705586	610	dbSNP
rs1489172240	614	dbSNP
rs780403203	616	dbSNP
rs1363540656	621	dbSNP
rs1265554343	628	dbSNP
rs1039022049	634	dbSNP
rs192351242	647	dbSNP
rs1305347023	650	dbSNP
rs1299401078	657	dbSNP
rs1231000809	667	dbSNP
rs769533768	683	dbSNP
rs909967844	698	dbSNP
rs777115522	716	dbSNP
rs1303313226	722	dbSNP
rs1296348315	730	dbSNP
rs1385636328	737	dbSNP
rs534673775	740	dbSNP
rs1030841907	742	dbSNP
rs1226581623	756	dbSNP
rs551815813	758	dbSNP
rs973233527	760	dbSNP
rs1170728218	764	dbSNP
rs1430149689	795	dbSNP
rs891914297	807	dbSNP
rs919087339	808	dbSNP
rs774368952	818	dbSNP
rs1052105571	821	dbSNP
rs1466263945	823	dbSNP
rs1267995725	825	dbSNP
rs1021819385	847	dbSNP
rs1479381501	855	dbSNP
rs1189298742	871	dbSNP
rs1276322296	874	dbSNP
rs1205517517	884	dbSNP
rs890152743	895	dbSNP
rs968928962	907	dbSNP
rs943049361	923	dbSNP
rs1231558709	934	dbSNP
rs1314417653	942	dbSNP
rs1302593986	946	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.