>CD81|NM_004356|3'UTR
   1 GGCCCCGCAGCTCTGGCCACAGGGACCTCTGCAGTGCCCCCTAAGTGACCCGGACACTTCCGAGGGGGCCATCACCGCCT
  81 GTGTATATAACGTTTCCGGTATTACTCTGCTACACGTAGCCTTTTTACTTTTGGGGTTTTGTTTTTGTTCTGAACTTTCC
 161 TGTTACCTTTTCAGGGCTGACGTCACATGTAGGTGGCGTGTATGAGTGGAGACGGGCCTGGGTCTTGGGGACTGGAGGGC
 241 AGGGGTCCTTCTGCCCTGGGGTCCCAGGGTGCTCTGCCTGCTCAGCCAGGCCTCTCCTGGGAGCCACTCGCCCAGAGACT
 321 CAGCTTGGCCAACTTGGGGGGCTGTGTCCACCCAGCCCGCCCGTCCTGTGGGCTGCACAGCTCACCTTGTTCCCTCCTGC
 401 CCCGGTTCGAGAGCCGAGTCTGTGGGCACTCTCTGCCTTCATGCACCTGTCCTTTCTAACACGTCGCCTTCAACTGTAAT
 481 CACAACATCCTGACTCCGTCATTTAATAAAGAAGGAACATCAGGCATGCTACCAGGCCTGTGCAGTCCCTCAG

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' aauaACGGGGAU----AAGAUUGUGu 5' ||| |||: ||||||||| Target 5' ttcaTGCACCTGTCCTTTCTAACACg 3'	438 - 463	164.00	-18.00
2	miRNA 3' aauAACGGGGAUAAGAUUGUGu 5' |||::::|:||||:| || Target 5' gttTTGTTTTTGTTCTGA-ACt 3'	136 - 156	107.00	-11.44
3	miRNA 3' aaUAAC-GGGGAU--A---AGAUUGUGu 5' | || |||||| | | :|||| Target 5' gcAGTGCCCCCTAAGTGACCCGGACACt 3'	31 - 58	106.00	-11.42

Mutant ID	Mutant Position	Mutant Source
COSN30454747	52	COSMIC
COSN26969920	53	COSMIC
COSN30498339	63	COSMIC
COSN20077370	71	COSMIC
COSN23655933	72	COSMIC
COSN30498128	101	COSMIC
COSN31526348	114	COSMIC
COSN24091553	364	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs774235346	2	dbSNP
rs1427506647	4	dbSNP
rs1417603458	5	dbSNP
rs948467244	6	dbSNP
rs188175983	7	dbSNP
rs368448552	8	dbSNP
rs755687318	9	dbSNP
rs1301767029	11	dbSNP
rs1365568283	12	dbSNP
rs765716594	13	dbSNP
rs1200811527	14	dbSNP
rs1451121769	16	dbSNP
rs753248682	23	dbSNP
rs1430743393	25	dbSNP
rs750214924	25	dbSNP
rs758755980	27	dbSNP
rs1389270135	28	dbSNP
rs1187893511	30	dbSNP
rs755947408	33	dbSNP
rs777957535	34	dbSNP
rs1241766781	36	dbSNP
rs747416444	38	dbSNP
rs757357708	40	dbSNP
rs779737723	40	dbSNP
rs10645	41	dbSNP
rs746329414	42	dbSNP
rs754474454	42	dbSNP
rs778297508	42	dbSNP
rs745930267	44	dbSNP
rs770043232	46	dbSNP
rs1206254690	50	dbSNP
rs775538716	52	dbSNP
rs748078366	53	dbSNP
rs911957710	54	dbSNP
rs1412830763	56	dbSNP
rs1340542723	57	dbSNP
rs1312238606	62	dbSNP
rs566859282	63	dbSNP
rs947280364	65	dbSNP
rs974738375	66	dbSNP
rs1039021328	69	dbSNP
rs1431366945	69	dbSNP
rs900539408	77	dbSNP
rs35800690	78	dbSNP
rs1458408410	84	dbSNP
rs1370125582	85	dbSNP
rs920586136	86	dbSNP
rs997454117	87	dbSNP
rs1239350293	88	dbSNP
rs74701050	92	dbSNP
rs1183219183	93	dbSNP
rs1484232153	96	dbSNP
rs1063316	98	dbSNP
rs1195588730	98	dbSNP
rs1210795616	99	dbSNP
rs886717708	107	dbSNP
rs950166251	112	dbSNP
rs1005470873	115	dbSNP
rs1354312291	116	dbSNP
rs1804493	117	dbSNP
rs963984605	121	dbSNP
rs1327861365	129	dbSNP
rs1348393882	129	dbSNP
rs905570764	132	dbSNP
rs1161426886	133	dbSNP
rs1459915143	135	dbSNP
rs1486948598	138	dbSNP
rs1175487757	148	dbSNP
rs1249948286	154	dbSNP
rs1001698574	155	dbSNP
rs1419842955	159	dbSNP
rs530580827	160	dbSNP
rs1365291795	161	dbSNP
rs113585763	165	dbSNP
rs1432698291	167	dbSNP
rs1268288819	168	dbSNP
rs892792063	178	dbSNP
rs1023882418	179	dbSNP
rs1375924788	182	dbSNP
rs955309220	183	dbSNP
rs1295901929	187	dbSNP
rs1440161092	189	dbSNP
rs986556099	198	dbSNP
rs749149940	199	dbSNP
rs143793173	201	dbSNP
rs1391583815	203	dbSNP
rs1308782797	210	dbSNP
rs1158252242	211	dbSNP
rs1369223718	214	dbSNP
rs928409359	215	dbSNP
rs975227943	220	dbSNP
rs1281500914	225	dbSNP
rs770452088	226	dbSNP
rs1049390	242	dbSNP
rs914380499	245	dbSNP
rs34939011	254	dbSNP
rs947206931	262	dbSNP
rs1210004076	266	dbSNP
rs1038951256	267	dbSNP
rs1444815321	270	dbSNP
rs1481021846	273	dbSNP
rs1283084045	274	dbSNP
rs1219500302	275	dbSNP
rs952010149	281	dbSNP
rs900424917	286	dbSNP
rs1251429331	293	dbSNP
rs1233304146	297	dbSNP
rs535211091	310	dbSNP
rs1454002635	311	dbSNP
rs908103201	323	dbSNP
rs933356773	328	dbSNP
rs1395486726	332	dbSNP
rs1051734494	333	dbSNP
rs1415327479	334	dbSNP
rs1049549	335	dbSNP
rs1353411824	336	dbSNP
rs1454125469	336	dbSNP
rs763029467	336	dbSNP
rs1191102395	337	dbSNP
rs568706017	342	dbSNP
rs1016858211	354	dbSNP
rs1474129850	355	dbSNP
rs927447082	358	dbSNP
rs899362364	359	dbSNP
rs535797531	360	dbSNP
rs767130888	361	dbSNP
rs557272729	363	dbSNP
rs1299398239	364	dbSNP
rs892915391	367	dbSNP
rs1023934673	369	dbSNP
rs1391744419	376	dbSNP
rs1439746294	385	dbSNP
rs1359948825	390	dbSNP
rs775196913	404	dbSNP
rs1373708133	407	dbSNP
rs575239537	409	dbSNP
rs1035338452	410	dbSNP
rs955993856	417	dbSNP
rs891008045	418	dbSNP
rs1008090176	419	dbSNP
rs1295576864	424	dbSNP
rs1308136288	431	dbSNP
rs1018228945	432	dbSNP
rs1419437905	434	dbSNP
rs988604325	437	dbSNP
rs1473509549	442	dbSNP
rs762386257	445	dbSNP
rs1186843322	447	dbSNP
rs770313718	452	dbSNP
rs995379058	452	dbSNP
rs1049648	457	dbSNP
rs138604169	459	dbSNP
rs1049652	463	dbSNP
rs183619205	464	dbSNP
rs750711443	466	dbSNP
rs1049659	467	dbSNP
rs907885191	468	dbSNP
rs933199989	474	dbSNP
rs1354420861	480	dbSNP
rs1184695753	482	dbSNP
rs971422598	484	dbSNP
rs1406576647	485	dbSNP
rs1415095732	498	dbSNP
rs541203101	499	dbSNP
rs559300491	527	dbSNP
rs1418636325	531	dbSNP
rs1052218780	532	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.