>CARD10|NM_014550|3'UTR
   1 GGCTCATCTGATACCTGCACCTTCTCCCCAAGCCAGCGTGGACCCTGGTGTCTATGGTGAAGCTGGGCCCTCCCACCCTG
  81 AGCCTTCCTAGACCCTTGGACTCTCAGATGCAGGGCCCTTGGCTCTGGCCTCTCACCCCCAAGGCTGTCTCTGGCCCTGC
 161 CGAGCCTATGGGAGTCCCGGGACAGAGTGCCCACTCCCCTCTACTTGCTGCTCTGGGCCTCCCCACCTTTCCTGGGGTCT
 241 CCACATTCCCACTAGTGGGTCTTATGTGTGTCTGTGTCTTCTCCTTAAACACTCGCCCTGGAGTCTGTTCTCACACCTGT
 321 GCGCAGGTTTGCACACTCAAGTTCTCATGGGCAGGCTCAGGTCTGTCCCGCTGCCCTGGGCACGAGGTCTCCTGAGGACC
 401 TGGGCCTGTTCTGCTCCTAGGAGACCTGAGCCCGTTACCGCGTGACTCCCACCATCCAGCTCGCGCTCCTCGTGGATTCA
 481 GCCATGCATGGACTGGGGTGTTCCCTGGCCCATGGTCACCTGTGCCCCTCGTGTCTCCTCACATGGGTGTCTGTGGTTCT
 561 CTCCTGTGTAAATGTCACGCCCCACCCCTGTTTCATGTGGGCACTAACACGTGTGCGTTCCTGGCGGGCACACTCAGGAC
 641 CGTGCCTCACAGGGCCCACTCCCTGCCTATGCCTCCCTCTTGGGGGGCCGAGGAGGGCGGCTGCTCTGTCATGAGAATGT
 721 ACGGCCCGTGGATGATTAACGGGCCTTTTTCACTTAGAAGCTGCACATTATGGAGCATTAAACACTTTTGTCATAGAAAA
 801 AAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gcgGCCCGAGGGGCCCc 5' :||| :|||||| Target 5' ctaTGGGAGTCCCGGGa 3'	166 - 182	134.00	-23.00
2	miRNA 3' gcGGCCCGAGGGGCCCc 5' || | : |||:||| Target 5' gtCCCGCTGCCCTGGGc 3'	365 - 381	131.00	-18.70
3	miRNA 3' gcGGCC---CGAGGGGCCcc 5' |:|| |:||||:|| Target 5' gaCTGGGGTGTTCCCTGGcc 3'	491 - 510	119.00	-20.50

Mutant ID	Mutant Position	Mutant Source
COSN8010483	19	COSMIC
COSN31595270	58	COSMIC
COSN30141037	71	COSMIC
COSN28879894	95	COSMIC
COSN24400825	189	COSMIC
COSN31488779	363	COSMIC
COSN30542638	391	COSMIC
COSN28142206	706	COSMIC
COSN9165642	759	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1376178288	2	dbSNP
rs963791706	7	dbSNP
rs913179053	15	dbSNP
rs907904309	21	dbSNP
rs1366121301	24	dbSNP
rs565287011	26	dbSNP
rs1182938028	28	dbSNP
rs1423641751	33	dbSNP
rs371369047	37	dbSNP
rs758830435	38	dbSNP
rs112729139	41	dbSNP
rs1157032457	44	dbSNP
rs959989611	46	dbSNP
rs374971945	47	dbSNP
rs1406250130	50	dbSNP
rs540594460	54	dbSNP
rs1325664022	56	dbSNP
rs1213897798	58	dbSNP
rs1352745585	59	dbSNP
rs1261282679	64	dbSNP
rs1035961945	67	dbSNP
rs1392856143	67	dbSNP
rs1338510918	68	dbSNP
rs1291628506	69	dbSNP
rs755738382	74	dbSNP
rs1237791021	77	dbSNP
rs1356110422	84	dbSNP
rs1358045526	85	dbSNP
rs181634983	87	dbSNP
rs772265562	88	dbSNP
rs1297647617	94	dbSNP
rs371102573	95	dbSNP
rs1387745237	96	dbSNP
rs1022190892	98	dbSNP
rs1251229843	104	dbSNP
rs11552597	116	dbSNP
rs542874338	124	dbSNP
rs1367089795	126	dbSNP
rs1000782838	135	dbSNP
rs1287982311	135	dbSNP
rs1189550117	136	dbSNP
rs867694425	138	dbSNP
rs1011366693	139	dbSNP
rs1262106673	140	dbSNP
rs1164605553	148	dbSNP
rs764205839	160	dbSNP
rs878866577	161	dbSNP
rs964216303	162	dbSNP
rs1467569705	169	dbSNP
rs1332630607	174	dbSNP
rs1052840946	176	dbSNP
rs1007853065	177	dbSNP
rs911397634	178	dbSNP
rs190162662	179	dbSNP
rs1049647013	185	dbSNP
rs1332393589	188	dbSNP
rs1220307990	192	dbSNP
rs955800044	196	dbSNP
rs1251748433	200	dbSNP
rs1025929740	207	dbSNP
rs556833221	228	dbSNP
rs994124691	245	dbSNP
rs932557959	251	dbSNP
rs538825026	254	dbSNP
rs962686168	257	dbSNP
rs1038603423	260	dbSNP
rs1486060416	261	dbSNP
rs1187461910	264	dbSNP
rs1016939143	265	dbSNP
rs1255761287	266	dbSNP
rs745925028	266	dbSNP
rs1478971199	274	dbSNP
rs939836135	276	dbSNP
rs571393550	278	dbSNP
rs1460947692	282	dbSNP
rs553018323	283	dbSNP
rs1174871035	287	dbSNP
rs983532013	294	dbSNP
rs534440808	295	dbSNP
rs905972404	296	dbSNP
rs1433552127	301	dbSNP
rs1269093180	303	dbSNP
rs1024547119	307	dbSNP
rs960039938	312	dbSNP
rs1480047597	314	dbSNP
rs1401553477	315	dbSNP
rs928605447	322	dbSNP
rs142420743	323	dbSNP
rs891706027	324	dbSNP
rs1053027058	328	dbSNP
rs1348686625	335	dbSNP
rs1320546590	337	dbSNP
rs1261661796	338	dbSNP
rs1021830829	340	dbSNP
rs1187432092	342	dbSNP
rs548583930	348	dbSNP
rs990734578	352	dbSNP
rs55735911	356	dbSNP
rs530287850	358	dbSNP
rs935619054	365	dbSNP
rs904203439	370	dbSNP
rs749473242	379	dbSNP
rs116707952	383	dbSNP
rs1376284424	389	dbSNP
rs1332057845	390	dbSNP
rs1434722095	399	dbSNP
rs911674585	405	dbSNP
rs1373189279	407	dbSNP
rs1455048708	412	dbSNP
rs1289125967	417	dbSNP
rs1360981874	423	dbSNP
rs1380368109	427	dbSNP
rs56404346	433	dbSNP
rs996753315	434	dbSNP
rs778133429	439	dbSNP
rs532727753	440	dbSNP
rs545701858	441	dbSNP
rs565369955	442	dbSNP
rs939866251	446	dbSNP
rs886915776	449	dbSNP
rs1212006477	453	dbSNP
rs368427540	462	dbSNP
rs375241158	464	dbSNP
rs1418096578	465	dbSNP
rs1392684286	470	dbSNP
rs1238263003	471	dbSNP
rs756412923	472	dbSNP
rs1179894306	484	dbSNP
rs1451288477	489	dbSNP
rs928657697	490	dbSNP
rs1395081583	491	dbSNP
rs1459585413	494	dbSNP
rs1024435334	503	dbSNP
rs1301222853	504	dbSNP
rs35209269	506	dbSNP
rs1375606922	507	dbSNP
rs1200358715	512	dbSNP
rs868519312	520	dbSNP
rs948717603	524	dbSNP
rs1315363497	526	dbSNP
rs1014466014	530	dbSNP
rs1346192220	533	dbSNP
rs1274232115	537	dbSNP
rs1468347691	540	dbSNP
rs186552334	561	dbSNP
rs41280029	568	dbSNP
rs1031564856	569	dbSNP
rs956417681	572	dbSNP
rs543048030	577	dbSNP
rs181295645	578	dbSNP
rs1038706467	579	dbSNP
rs1353175440	580	dbSNP
rs563591886	587	dbSNP
rs557069898	590	dbSNP
rs527939113	592	dbSNP
rs1166698659	603	dbSNP
rs1449168944	604	dbSNP
rs377188158	610	dbSNP
rs1380647248	611	dbSNP
rs1333474335	614	dbSNP
rs372977739	616	dbSNP
rs1404111174	617	dbSNP
rs1442945033	620	dbSNP
rs1297702420	622	dbSNP
rs147337306	623	dbSNP
rs1235330775	624	dbSNP
rs1051522427	625	dbSNP
rs996470904	626	dbSNP
rs1231913564	629	dbSNP
rs898136022	632	dbSNP
rs1308624592	637	dbSNP
rs1204564105	640	dbSNP
rs55992715	640	dbSNP
rs1004118406	641	dbSNP
rs879907267	642	dbSNP
rs972933530	644	dbSNP
rs1474720700	647	dbSNP
rs765298639	648	dbSNP
rs1056305923	655	dbSNP
rs546896109	658	dbSNP
rs1187492113	663	dbSNP
rs1169703803	668	dbSNP
rs56057017	668	dbSNP
rs5750441	669	dbSNP
rs970088060	675	dbSNP
rs770908711	676	dbSNP
rs1272325483	679	dbSNP
rs1413273554	679	dbSNP
rs188097898	683	dbSNP
rs1367173743	685	dbSNP
rs1214477628	688	dbSNP
rs569589077	689	dbSNP
rs1242542394	690	dbSNP
rs955835752	691	dbSNP
rs56174981	698	dbSNP
rs1280526159	699	dbSNP
rs1485152787	711	dbSNP
rs1185479934	713	dbSNP
rs144107916	722	dbSNP
rs765918886	723	dbSNP
rs571780934	727	dbSNP
rs139823002	728	dbSNP
rs183520474	740	dbSNP
rs890133124	741	dbSNP
rs1051920018	742	dbSNP
rs200239551	743	dbSNP
rs986922141	751	dbSNP
rs56094714	757	dbSNP
rs897609920	764	dbSNP
rs561366710	767	dbSNP
rs549541475	769	dbSNP
rs1268868635	772	dbSNP
rs530920156	774	dbSNP
rs1235788197	785	dbSNP
rs1303544523	787	dbSNP
rs955500116	790	dbSNP
rs879369730	793	dbSNP
rs975159964	794	dbSNP
rs3180586	796	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.