>CCL11|NM_002986|3'UTR
   1 ATAATCACCATTTTTGAAACCAAACCAGAGCCTGAGTGTTGCCTAATTTGTTTTCCCTTCTTACAATGCATTCTGAGGTA
  81 ACCTCATTATCAGTCCAAAGGGCATGGGTTTTATTATATATATATATTTTTTTTTTTAAAAAAAAAACGTATTGCATTTA
 161 ATTTATTGAGGCTTTAAAACTTATCCTCCATGAATATCAGTTATTTTTAAACTGTAAAGCTTTGTGCAGATTCTTTACCC
 241 CCTGGGAGCCCCAATTCGATCCCCTGTCACGTGTGGGCAATGTTCCCCCTCTCCTCTCTTCCTCCCTGGAATCTTGTAAA
 321 GGTCCTGGCAAAGATGATCAGTATGAAAATGTCATTGTTCTTGTGAACCCAAAGTGTGACTCATTAAATGGAAGTAAATG
 401 TTGTTTTAGGAATACATAAAGTATGTGCATATTTTATTATAGTCACTAGTTGTAATTTTTTTGTGGGAAATCCACACTGA
 481 GCTGAGGGGG

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gaguGUGUGACAGAGGGACCu 5' | | || |||||||| Target 5' tctcCTCTCTTCCTCCCTGGa 3'	290 - 310	153.00	-22.50
2	miRNA 3' gaGUGUGUGACA-GAGGGACCu 5' || |: || | | |||||| Target 5' tgCAGATTCTTTACCCCCTGGg 3'	225 - 246	127.00	-14.50
3	miRNA 3' gaGUGUGUGAC-AGAGGGAccu 5' | :| :|| |:||||| Target 5' tgCCTAATTTGTTTTCCCTtct 3'	40 - 61	111.00	-8.02

Mutant ID	Mutant Position	Mutant Source
COSM5548197	2	COSMIC
COSN30456601	9	COSMIC
COSN30511547	10	COSMIC
COSN30129810	31	COSMIC
COSN30511495	32	COSMIC
COSN31613229	35	COSMIC
COSN31557782	47	COSMIC
COSN20079068	70	COSMIC
COSN31607745	83	COSMIC
COSN15661001	87	COSMIC
COSN31561469	123	COSMIC
COSN23301304	127	COSMIC
COSN20082008	128	COSMIC
COSN15664459	129	COSMIC
COSN30626905	130	COSMIC
COSN7164885	130	COSMIC
COSN30531760	131	COSMIC
COSN17648507	133	COSMIC
COSN15663819	137	COSMIC
COSN20079131	137	COSMIC
COSN17410072	138	COSMIC
COSN28183514	138	COSMIC
COSN28866460	138	COSMIC
COSN28196159	139	COSMIC
COSN30116959	140	COSMIC
COSN15665026	142	COSMIC
COSN30751047	143	COSMIC
COSN16967483	150	COSMIC
COSN29103683	463	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1463049827	2	dbSNP
rs973714630	3	dbSNP
rs1318573623	5	dbSNP
rs1387842743	7	dbSNP
rs200831316	11	dbSNP
rs748251984	17	dbSNP
rs758511524	21	dbSNP
rs762752655	22	dbSNP
rs1293508472	26	dbSNP
rs557784509	27	dbSNP
rs201621608	30	dbSNP
rs769693180	32	dbSNP
rs200369153	35	dbSNP
rs1206233877	37	dbSNP
rs768733712	38	dbSNP
rs773956209	39	dbSNP
rs761411782	40	dbSNP
rs1418884920	46	dbSNP
rs767017905	48	dbSNP
rs201160159	56	dbSNP
rs1180084973	62	dbSNP
rs1482806563	67	dbSNP
rs1291177845	75	dbSNP
rs1238737829	82	dbSNP
rs920978933	83	dbSNP
rs199763228	86	dbSNP
rs1200390881	90	dbSNP
rs1354626792	92	dbSNP
rs1265257512	100	dbSNP
rs1429817680	104	dbSNP
rs1434653947	105	dbSNP
rs1173923537	106	dbSNP
rs1358526615	108	dbSNP
rs1462397175	114	dbSNP
rs1334705404	115	dbSNP
rs200454183	115	dbSNP
rs200374644	117	dbSNP
rs1388472023	120	dbSNP
rs201428314	122	dbSNP
rs1163961146	123	dbSNP
rs149027038	123	dbSNP
rs966410792	123	dbSNP
rs1408903071	124	dbSNP
rs1201590952	125	dbSNP
rs1283524422	125	dbSNP
rs1469670590	125	dbSNP
rs201263708	126	dbSNP
rs1329259337	127	dbSNP
rs1331741238	127	dbSNP
rs1349280175	127	dbSNP
rs1438446497	127	dbSNP
rs1491463706	127	dbSNP
rs201481266	127	dbSNP
rs201654151	128	dbSNP
rs5027581	129	dbSNP
rs71784956	129	dbSNP
rs200850991	130	dbSNP
rs768719395	131	dbSNP
rs1308636136	132	dbSNP
rs979082222	133	dbSNP
rs924961777	134	dbSNP
rs1189823481	135	dbSNP
rs1466516560	135	dbSNP
rs1206395871	136	dbSNP
rs367665833	136	dbSNP
rs376201601	136	dbSNP
rs1019111	137	dbSNP
rs1302556261	137	dbSNP
rs1491203445	137	dbSNP
rs371836921	137	dbSNP
rs1019110	138	dbSNP
rs1169733778	138	dbSNP
rs1336475721	138	dbSNP
rs1359952624	138	dbSNP
rs1374979657	138	dbSNP
rs1412804699	138	dbSNP
rs1491394429	138	dbSNP
rs35429363	138	dbSNP
rs397723282	138	dbSNP
rs76028040	139	dbSNP
rs201906937	140	dbSNP
rs957883933	141	dbSNP
rs1237658615	142	dbSNP
rs1211857700	143	dbSNP
rs991745040	144	dbSNP
rs1205699065	146	dbSNP
rs916112994	146	dbSNP
rs1344242533	147	dbSNP
rs950247208	147	dbSNP
rs1213771436	148	dbSNP
rs200124756	149	dbSNP
rs929361485	149	dbSNP
rs201073234	150	dbSNP
rs1424157388	171	dbSNP
rs1308721086	173	dbSNP
rs551139066	176	dbSNP
rs887781759	184	dbSNP
rs1170388554	185	dbSNP
rs1406295344	190	dbSNP
rs201817342	202	dbSNP
rs912377770	209	dbSNP
rs1303438758	212	dbSNP
rs200100094	213	dbSNP
rs934521776	219	dbSNP
rs901718804	221	dbSNP
rs191484864	228	dbSNP
rs200912705	230	dbSNP
rs1249513793	235	dbSNP
rs1203794319	237	dbSNP
rs201751198	239	dbSNP
rs200274578	241	dbSNP
rs1000554042	242	dbSNP
rs1032464937	245	dbSNP
rs1231314263	249	dbSNP
rs530277159	254	dbSNP
rs111976983	255	dbSNP
rs41362349	258	dbSNP
rs202172123	259	dbSNP
rs1461302269	263	dbSNP
rs1293959795	264	dbSNP
rs1002938109	271	dbSNP
rs200523282	272	dbSNP
rs1473275710	277	dbSNP
rs1245961040	278	dbSNP
rs1247834223	282	dbSNP
rs1486053442	287	dbSNP
rs760900461	288	dbSNP
rs566855110	290	dbSNP
rs201386262	292	dbSNP
rs1488770682	297	dbSNP
rs199525457	301	dbSNP
rs200522301	303	dbSNP
rs886604300	306	dbSNP
rs201540202	307	dbSNP
rs1268977156	312	dbSNP
rs1230484271	316	dbSNP
rs199726701	323	dbSNP
rs200687580	329	dbSNP
rs1220258606	334	dbSNP
rs201665847	337	dbSNP
rs1163667407	339	dbSNP
rs1440591521	341	dbSNP
rs199739461	357	dbSNP
rs909255532	359	dbSNP
rs1415277396	364	dbSNP
rs943387499	369	dbSNP
rs1403300028	375	dbSNP
rs1157754791	386	dbSNP
rs200846588	411	dbSNP
rs1019109	412	dbSNP
rs933227622	422	dbSNP
rs374029930	423	dbSNP
rs1467825362	424	dbSNP
rs199863373	431	dbSNP
rs912339861	432	dbSNP
rs568943172	438	dbSNP
rs1248441753	441	dbSNP
rs542500214	451	dbSNP
rs1485022424	452	dbSNP
rs1260419499	456	dbSNP
rs1314062540	456	dbSNP
rs902355715	456	dbSNP
rs1301853104	459	dbSNP
rs41274294	463	dbSNP
rs1366330615	465	dbSNP
rs1300276500	468	dbSNP
rs569292127	483	dbSNP
rs1032092706	489	dbSNP
rs914463957	492	dbSNP
rs1466081501	498	dbSNP
rs947302951	499	dbSNP
rs1044356826	501	dbSNP
rs201599560	507	dbSNP
rs938700454	510	dbSNP
rs1025047584	512	dbSNP
rs1190254404	516	dbSNP
rs1057237536	528	dbSNP
rs1261259403	530	dbSNP
rs886528327	531	dbSNP
rs1182713240	532	dbSNP
rs1005020240	537	dbSNP
rs971662836	538	dbSNP
rs1285211542	539	dbSNP
rs1329676118	540	dbSNP
rs981687170	541	dbSNP
rs1016858245	542	dbSNP
rs1026518413	543	dbSNP
rs557017215	546	dbSNP
rs996367708	557	dbSNP
rs1326254896	558	dbSNP
rs985325705	572	dbSNP
rs1396276893	573	dbSNP
rs1029229841	580	dbSNP
rs965129562	585	dbSNP
rs953710350	588	dbSNP
rs986431590	592	dbSNP
rs1433620336	593	dbSNP
rs1019326550	597	dbSNP
rs1371082830	601	dbSNP
rs1170455582	602	dbSNP
rs1238746825	606	dbSNP
rs974801858	615	dbSNP
rs765800738	616	dbSNP
rs1180985818	617	dbSNP
rs1408287128	618	dbSNP
rs1458429189	621	dbSNP
rs1255422257	626	dbSNP
rs1187297136	628	dbSNP
rs184861390	635	dbSNP
rs1157443425	636	dbSNP
rs955996869	638	dbSNP
rs753641967	643	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.