>MEMO1|NM_001137602|3'UTR
   1 AGCTCTGAATCCTCAGGGATGCCACCTGCACATTCTCATACTCTGTCCGGGGTCCCAGCCTAGCCTTTACCACGATACTG
  81 GTCCTGGTTTGGGGGGATTCTGAAACCTCAAACTAATAGAACTTTCTTCTCTTTTTTTCTAGTAGGTGTAGTCCTTCCTT
 161 AATTTCAACTCATTAAAAAATGCTTTATAGTTTAGGGCAGTGGAAGGAAGGCTGGCATCAAAATATTTTGATCAAAAAAG
 241 ATGACAATGTAAAGGCTCAGTTGTGGCAGACAGTTTTTTGAAAGTAACTTGTAAAGCATTTACCATATCCTAAATTTGCA
 321 CTCTTTGCAGACTTGTGCACATATATTCCGCTTTCAGAATAGTTTTGCAAATTGTACACAAACAAACAAAAAGGTGGAAG
 401 CTTTTTAATAAAGAAATTGCATTTATAAATGATCTGTATTAGAATATAATAAATCTCCAGTTATAGTCAATTACTACCCA
 481 TGTTGTACAACAGATACCTTCTATTTTAGTTGCTAATAAAGGGCTACACAACTCAAAATAGTCTGATTTAAAAAAAAAAA
 561 AAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gguagaaggaaGUCGGAg 5' |||||| Target 5' gtccggggtccCAGCCTa 3'	45 - 62	120.00	-15.80
2	miRNA 3' ggUAGAAGG---AAGUCggag 5' || |||| ||||| Target 5' atATATTCCGCTTTCAGaata 3'	341 - 361	100.00	-8.56
3	miRNA 3' ggUAGA----AGGAA-GUCGGAg 5' ||:| ||:|| ||| || Target 5' aaATTTGCACTCTTTGCAGACTt 3'	312 - 334	94.00	-6.70

Mutant ID	Mutant Position	Mutant Source
COSN30101110	48	COSMIC
COSN31612952	60	COSMIC
COSN31506750	73	COSMIC
COSN29467437	202	COSMIC
COSN28813924	296	COSMIC
COSN30176481	350	COSMIC
COSN31489932	534	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1374233804	2	dbSNP
rs1462782715	5	dbSNP
rs1168227475	7	dbSNP
rs751927794	11	dbSNP
rs1410092428	14	dbSNP
rs764813797	18	dbSNP
rs756928464	21	dbSNP
rs753472562	22	dbSNP
rs1001832867	23	dbSNP
rs1299882000	28	dbSNP
rs1376263734	33	dbSNP
rs1257985858	37	dbSNP
rs376717212	40	dbSNP
rs763637397	42	dbSNP
rs764131957	44	dbSNP
rs760125759	47	dbSNP
rs144466744	48	dbSNP
rs767562663	49	dbSNP
rs1336540624	51	dbSNP
rs1014263878	52	dbSNP
rs1216455403	59	dbSNP
rs886475625	63	dbSNP
rs532002978	73	dbSNP
rs1308507998	74	dbSNP
rs1207243094	79	dbSNP
rs1047832026	84	dbSNP
rs1482938718	87	dbSNP
rs1188786845	97	dbSNP
rs1259749562	97	dbSNP
rs1476949288	101	dbSNP
rs930739062	117	dbSNP
rs1418519024	119	dbSNP
rs1245484475	122	dbSNP
rs879006531	126	dbSNP
rs1316265957	130	dbSNP
rs1172376833	139	dbSNP
rs562978376	145	dbSNP
rs190783152	168	dbSNP
rs1312928894	169	dbSNP
rs1039216400	171	dbSNP
rs943584441	172	dbSNP
rs1397737386	181	dbSNP
rs939525365	181	dbSNP
rs912148257	184	dbSNP
rs928197282	187	dbSNP
rs1312087899	188	dbSNP
rs186930444	191	dbSNP
rs988167657	198	dbSNP
rs865842479	206	dbSNP
rs1022405246	211	dbSNP
rs1475592147	216	dbSNP
rs933649071	218	dbSNP
rs1188084611	220	dbSNP
rs989235137	224	dbSNP
rs554457452	227	dbSNP
rs1453706718	230	dbSNP
rs953891368	247	dbSNP
rs1197452896	248	dbSNP
rs541228601	249	dbSNP
rs1345448701	251	dbSNP
rs1393028236	254	dbSNP
rs1432504486	258	dbSNP
rs1273417045	271	dbSNP
rs1235660851	274	dbSNP
rs1367415287	274	dbSNP
rs1306273063	280	dbSNP
rs1351524003	288	dbSNP
rs573326541	290	dbSNP
rs1285170382	293	dbSNP
rs1453755413	296	dbSNP
rs997992694	305	dbSNP
rs1244756044	308	dbSNP
rs35697513	310	dbSNP
rs1343936067	313	dbSNP
rs1194114241	315	dbSNP
rs1396830146	319	dbSNP
rs1437231807	328	dbSNP
rs62142066	330	dbSNP
rs1157447157	332	dbSNP
rs1408135296	336	dbSNP
rs553522483	344	dbSNP
rs957135949	349	dbSNP
rs1394118154	351	dbSNP
rs1442115011	358	dbSNP
rs1473125221	362	dbSNP
rs1375372224	363	dbSNP
rs1015502914	366	dbSNP
rs1033228688	370	dbSNP
rs1353011460	374	dbSNP
rs1226822209	383	dbSNP
rs886432980	386	dbSNP
rs1428977304	391	dbSNP
rs979963772	391	dbSNP
rs372513207	393	dbSNP
rs970335977	410	dbSNP
rs1456365194	413	dbSNP
rs994850187	417	dbSNP
rs1024219612	421	dbSNP
rs1470881350	422	dbSNP
rs1161035552	431	dbSNP
rs1055228194	434	dbSNP
rs939497522	455	dbSNP
rs539806594	463	dbSNP
rs1014211606	464	dbSNP
rs886421746	469	dbSNP
rs1042603811	470	dbSNP
rs945663980	478	dbSNP
rs914907281	485	dbSNP
rs989519243	493	dbSNP
rs1235418456	515	dbSNP
rs953753157	528	dbSNP
rs1274794672	530	dbSNP
rs920991188	534	dbSNP
rs1026337346	539	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.