>MTCH1|NM_014341|3'UTR
   1 CCTGAATCATCTAAAAAACACGGTCTCAACCTGGCCACCGTGGGTGAGGCCTGACCACCTTGGGACACCTGCAAGACGAC
  81 TCCAACCCAACAACAACCAGATGTGCTCCAGCCCAGCCGGGCTTCAGTTCCATATTTGCCATGTGTCTGTCCAGATGTGG
 161 GGTTGAGCGGGGGTGGGGCTGCACCCAGTGGATTGGGTCACCCGGCAGACCTAGGGAAGGTGAGGCGAGGTGGGGAGTTG
 241 GCAGAATCCCCATACCTCGCAGATTTGCTGAGTCTGTCTTGTGCAGAGGGCCAGAGAATGGCTTATGGGGGCCCAGGTTG
 321 GATGGGGAAAGGCTAATGGGGTCAGACCCCACCCCGTCTACCCCTCCAGTCAGCCCAGCGCCCATCCTGCAGCTCAGCTG
 401 GGAGCATCATTCTCCTGCTTTGTACATAGGGTGTGGTCCCCTGGCACGTGGCCACCATCATGTCTAGGCCTATGCTAGGA
 481 GGCAAATGGCCAGGCTCTGCCTGTGTTTTTCTCAACACTACTTTTCTGATATGAGGGCAGCACCTGCCTCTGAATGGGAA
 561 ATCATGCAACTACTCAGAATGTGTCCTCCTCATCTAATGCTCATCTGTTTAATGGTGATGCCTCGCGTACAGGATCTGGT
 641 TACCTGTGCAGTTGTGAATACCCAGAGGTTGGGCAGATCAGTGTCTCTAGTCCTACCCAGTTTTAAAGTTCATGGTAAGA
 721 TTTGACCTCATCTCCCGCAAATAAATGTATTGGTGATTTGGAGTTTTTAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gaucGGAG--GUACU--UAAGACUAc 5' :||| || | ||||||| Target 5' ttttTCTCAACACTACTTTTCTGATa 3'	506 - 531	142.00	-7.29
2	miRNA 3' gauCGGAGGUACUUAAGACUAc 5' || | || || ||||:| Target 5' ctcGCGTACAGGA--TCTGGTt 3'	622 - 641	114.00	-8.20
3	miRNA 3' gaucGGAG-GUACUUAA-GACUac 5' |||| || || || |||| Target 5' cataCCTCGCA-GATTTGCTGAgt 3'	251 - 273	109.00	-11.40

Mutant ID	Mutant Position	Mutant Source
COSM6716173	4	COSMIC
COSM6344935	8	COSMIC
COSM6716181	12	COSMIC
COSM8967570	21	COSMIC
COSM9560939	39	COSMIC
COSM8081460	46	COSMIC
COSM8841079	55	COSMIC
COSM3949134	59	COSMIC
COSM8952663	77	COSMIC
COSM7824157	88	COSMIC
COSM9060291	105	COSMIC
COSN31611018	281	COSMIC
COSN30539123	373	COSMIC
COSN30539125	374	COSMIC
COSN26649795	501	COSMIC
COSN15758064	586	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs113835599	1	dbSNP
rs548014532	8	dbSNP
rs777224461	9	dbSNP
rs771554982	10	dbSNP
rs778311595	13	dbSNP
rs752508381	19	dbSNP
rs780830612	21	dbSNP
rs773313267	22	dbSNP
rs1010568918	23	dbSNP
rs1433358658	25	dbSNP
rs202104211	27	dbSNP
rs1486846189	30	dbSNP
rs748349904	37	dbSNP
rs1042987	39	dbSNP
rs745677060	40	dbSNP
rs368953116	44	dbSNP
rs1184622981	46	dbSNP
rs757181411	48	dbSNP
rs751490722	49	dbSNP
rs375927561	51	dbSNP
rs757879436	52	dbSNP
rs1373794039	56	dbSNP
rs1192763003	58	dbSNP
rs1487462180	60	dbSNP
rs752328534	61	dbSNP
rs764996030	64	dbSNP
rs1485616750	65	dbSNP
rs754739268	69	dbSNP
rs1277161986	70	dbSNP
rs1216627959	71	dbSNP
rs754253049	72	dbSNP
rs697750	73	dbSNP
rs1216410192	74	dbSNP
rs761239610	77	dbSNP
rs535364523	78	dbSNP
rs765140134	81	dbSNP
rs138282989	83	dbSNP
rs1017973579	87	dbSNP
rs758397722	89	dbSNP
rs1330815241	90	dbSNP
rs761799358	91	dbSNP
rs181226085	98	dbSNP
rs188810107	100	dbSNP
rs571265988	103	dbSNP
rs759460025	103	dbSNP
rs201755942	108	dbSNP
rs776194400	111	dbSNP
rs570489883	112	dbSNP
rs770735930	113	dbSNP
rs746927170	116	dbSNP
rs946855249	118	dbSNP
rs777576922	119	dbSNP
rs757867804	125	dbSNP
rs550643684	127	dbSNP
rs537092263	130	dbSNP
rs1293560957	140	dbSNP
rs778637659	143	dbSNP
rs754649239	145	dbSNP
rs939661394	146	dbSNP
rs753596466	149	dbSNP
rs1256973969	151	dbSNP
rs1442826057	161	dbSNP
rs1213958229	162	dbSNP
rs376203471	163	dbSNP
rs756463953	168	dbSNP
rs551497213	169	dbSNP
rs1396882690	170	dbSNP
rs989169407	173	dbSNP
rs750462049	178	dbSNP
rs1411718602	184	dbSNP
rs1296506688	185	dbSNP
rs1179067778	186	dbSNP
rs1028376722	187	dbSNP
rs1470786459	189	dbSNP
rs548164971	192	dbSNP
rs909945712	195	dbSNP
rs11548580	196	dbSNP
rs901047778	199	dbSNP
rs1470109388	202	dbSNP
rs560808197	203	dbSNP
rs546904388	204	dbSNP
rs1254902799	216	dbSNP
rs761994133	226	dbSNP
rs145648761	227	dbSNP
rs1018610583	232	dbSNP
rs185224479	234	dbSNP
rs562174529	249	dbSNP
rs1430545918	252	dbSNP
rs1377948850	255	dbSNP
rs544295421	258	dbSNP
rs894495445	259	dbSNP
rs1457295682	260	dbSNP
rs530865782	261	dbSNP
rs938952845	267	dbSNP
rs1397989743	278	dbSNP
rs561761900	279	dbSNP
rs1441063141	289	dbSNP
rs1319441150	294	dbSNP
rs1359364578	296	dbSNP
rs1383590045	305	dbSNP
rs928871666	305	dbSNP
rs977654873	311	dbSNP
rs946176843	313	dbSNP
rs1021315406	318	dbSNP
rs1275673334	321	dbSNP
rs1335516519	347	dbSNP
rs1208133590	352	dbSNP
rs1457958771	353	dbSNP
rs1266352305	355	dbSNP
rs913681984	356	dbSNP
rs989241523	357	dbSNP
rs1262155535	358	dbSNP
rs1009963748	367	dbSNP
rs1423101705	367	dbSNP
rs1441105068	368	dbSNP
rs1461910885	372	dbSNP
rs1157716646	379	dbSNP
rs566635208	380	dbSNP
rs1454972674	381	dbSNP
rs1238440020	383	dbSNP
rs1027993205	389	dbSNP
rs1378896943	390	dbSNP
rs1413365840	391	dbSNP
rs892634646	407	dbSNP
rs1178377114	412	dbSNP
rs1456753217	413	dbSNP
rs1304887956	422	dbSNP
rs1318870077	424	dbSNP
rs180930776	426	dbSNP
rs543693371	427	dbSNP
rs1255667567	429	dbSNP
rs1052652942	430	dbSNP
rs965358603	431	dbSNP
rs1259396111	435	dbSNP
rs1211027065	447	dbSNP
rs1019947704	448	dbSNP
rs1405149581	452	dbSNP
rs1420427043	457	dbSNP
rs1159340542	465	dbSNP
rs11548582	468	dbSNP
rs772454726	469	dbSNP
rs1315096908	471	dbSNP
rs1376457146	483	dbSNP
rs1045745313	485	dbSNP
rs948051847	496	dbSNP
rs1242438463	502	dbSNP
rs878867571	507	dbSNP
rs1043176	526	dbSNP
rs11841	531	dbSNP
rs760149273	532	dbSNP
rs1354639442	537	dbSNP
rs1048506268	542	dbSNP
rs930078334	544	dbSNP
rs553218588	551	dbSNP
rs1251329927	552	dbSNP
rs1345948448	557	dbSNP
rs1236779558	561	dbSNP
rs918681987	565	dbSNP
rs976572729	567	dbSNP
rs1003280492	570	dbSNP
rs907473290	580	dbSNP
rs1481315926	590	dbSNP
rs1043214	594	dbSNP
rs190478682	598	dbSNP
rs1177234923	605	dbSNP
rs1423859544	608	dbSNP
rs1452120784	617	dbSNP
rs965604286	621	dbSNP
rs185570556	624	dbSNP
rs911110389	625	dbSNP
rs370748224	626	dbSNP
rs968335393	627	dbSNP
rs1443607792	647	dbSNP
rs1021286266	650	dbSNP
rs1389941690	657	dbSNP
rs1009935840	667	dbSNP
rs955757434	670	dbSNP
rs556834916	680	dbSNP
rs1421030473	695	dbSNP
rs1443364886	697	dbSNP
rs1304532900	700	dbSNP
rs1213826823	712	dbSNP
rs776942418	712	dbSNP
rs769011744	713	dbSNP
rs1003691369	723	dbSNP
rs1205152575	726	dbSNP
rs1379663624	729	dbSNP
rs756417239	734	dbSNP
rs1253288619	736	dbSNP
rs1474887981	737	dbSNP
rs1166480279	765	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.