>WNT10B|NM_003394|3'UTR
   1 GGGTCAGCCTTACCTTGGGGCTGGGGAAGAGGACTGTGTGAGAGGGGCGCCTTTTCAGCCCTTTGCTCTGATTTCCTTCC
  81 AAGGTCACTCTTGGTCCCTGGAAGCTTAAAGTATCTACCTGGAAACAGCTTTAGGGGTGGTGGGGGTCAGGTGGACTCTG
 161 GGATGTGTAGCCTTCTCCCCAACAATTGGAGGGTCTTGAGGGGAAGCTGCCACCCCTCTTCTGCTCCTTAGACACCTGAA
 241 TGGACTAAGATGAAATGCACTGTATTGCTCCTCCCACTTCTCAACTCCAGAGCCCCTTTAACCCTGATTCATACTCCTTT
 321 TGGCTGGGGAGTCCCTATAGTTTCACCACTCCTCTCCCTTGAGGGATAACCCCAGGCACTGTTTGGAGCCATAAGATCTG
 401 TATCTAGAAAGAGATCACCCACTCCTATGTACTATCCCCAAACTCCTTTACTGCAGCCTGGGCTCCCTCTTGTGGGATAA
 481 TGGGAGACAGTGGTAGAGAGGTTTTTCTTGGGAAAGAGACAGAGTGCTGAGGGGCACTCTCCCCTGAATCCTCAGAGAGT
 561 TGTCTGTCCAGGCCCTTAGGGAAGTTGTCTCCTTCCATTCAGATGTTAATGGGGACCCTCCAAAGGAAGGGGTTTTCCCA
 641 TGACTCTTGGAGCCTCTTTTTCCTTCTTCAGCAGGAAGGGTGGGAAGGGATAATTTATCATACTGAGACTTGTTCTTGGT
 721 TCCTGTTTGAAACTAAAATAAATTAAGTTACTGGAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' acccugagguacGGGGUUc 5' |||||| Target 5' gtgtagccttctCCCCAAc 3'	165 - 183	120.00	-10.80
2	miRNA 3' acccugagguacGGGGUUc 5' |||||| Target 5' cctatgtactatCCCCAAa 3'	424 - 442	120.00	-11.20
3	miRNA 3' acccUGAGGU-ACGGGGuuc 5' |||||| ||||| Target 5' ctcaACTCCAGAGCCCCttt 3'	280 - 299	119.00	-17.12

Mutant ID	Mutant Position	Mutant Source
COSN31540097	1	COSMIC
COSN30162714	5	COSMIC
COSN30513733	23	COSMIC
COSN20882101	29	COSMIC
COSN30462007	29	COSMIC
COSN31587462	31	COSMIC
COSN30148389	48	COSMIC
COSN30461414	51	COSMIC
COSN30170338	56	COSMIC
COSN9562376	142	COSMIC
COSN30529705	144	COSMIC
COSN24911201	177	COSMIC
COSN28726972	202	COSMIC
COSN23559180	235	COSMIC
COSN31561801	264	COSMIC
COSN28680688	339	COSMIC
COSN17183132	404	COSMIC
COSN7616434	421	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1310767071	3	dbSNP
rs779451842	4	dbSNP
rs1230495356	15	dbSNP
rs755470432	18	dbSNP
rs1324135436	20	dbSNP
rs754202358	22	dbSNP
rs1358418753	25	dbSNP
rs766668613	26	dbSNP
rs1420506374	28	dbSNP
rs1453514007	41	dbSNP
rs372083015	48	dbSNP
rs750617702	49	dbSNP
rs763131979	64	dbSNP
rs1045036279	79	dbSNP
rs189755508	84	dbSNP
rs1353269532	88	dbSNP
rs1263066392	99	dbSNP
rs1412284675	104	dbSNP
rs897548935	107	dbSNP
rs1018723572	119	dbSNP
rs1302671797	134	dbSNP
rs949415362	137	dbSNP
rs896554764	142	dbSNP
rs1182071166	160	dbSNP
rs888789026	164	dbSNP
rs1476922799	167	dbSNP
rs1171743194	171	dbSNP
rs1464836977	174	dbSNP
rs1375592125	175	dbSNP
rs1049972554	177	dbSNP
rs1470903438	182	dbSNP
rs77211836	185	dbSNP
rs1398196899	197	dbSNP
rs879134184	200	dbSNP
rs929794951	214	dbSNP
rs1329805538	228	dbSNP
rs1337688212	233	dbSNP
rs1231565125	241	dbSNP
rs919759963	249	dbSNP
rs1179383653	251	dbSNP
rs1324939952	252	dbSNP
rs531562481	254	dbSNP
rs775673343	262	dbSNP
rs1255674163	266	dbSNP
rs1338201928	267	dbSNP
rs942593669	268	dbSNP
rs1484018751	273	dbSNP
rs1251128482	275	dbSNP
rs3741627	291	dbSNP
rs945594772	295	dbSNP
rs756676096	297	dbSNP
rs986453506	302	dbSNP
rs913946302	315	dbSNP
rs1372373253	318	dbSNP
rs1461547313	334	dbSNP
rs992273706	335	dbSNP
rs1398932556	337	dbSNP
rs552152957	338	dbSNP
rs1328731388	339	dbSNP
rs781705638	342	dbSNP
rs1346532755	346	dbSNP
rs1020022475	360	dbSNP
rs771269925	369	dbSNP
rs1227553188	376	dbSNP
rs980771967	379	dbSNP
rs1353871066	389	dbSNP
rs532458319	399	dbSNP
rs747364437	401	dbSNP
rs184464227	416	dbSNP
rs1490783238	422	dbSNP
rs956861967	424	dbSNP
rs951960430	425	dbSNP
rs920545025	427	dbSNP
rs541034659	428	dbSNP
rs1001074244	436	dbSNP
rs961915667	441	dbSNP
rs1404718791	446	dbSNP
rs1163196401	449	dbSNP
rs1019073766	466	dbSNP
rs1426985018	467	dbSNP
rs1304241644	470	dbSNP
rs905438685	472	dbSNP
rs1384936881	477	dbSNP
rs1023864729	480	dbSNP
rs1383239789	488	dbSNP
rs1470049775	490	dbSNP
rs1387414953	501	dbSNP
rs1338178889	507	dbSNP
rs1244527078	516	dbSNP
rs1315314161	521	dbSNP
rs757105532	524	dbSNP
rs777418277	524	dbSNP
rs896502575	526	dbSNP
rs1156379533	531	dbSNP
rs1473257897	540	dbSNP
rs1486174434	545	dbSNP
rs1206816164	554	dbSNP
rs1047106152	556	dbSNP
rs1264015420	560	dbSNP
rs1442865400	563	dbSNP
rs1364340970	568	dbSNP
rs1186667056	573	dbSNP
rs748104831	574	dbSNP
rs953143732	575	dbSNP
rs1421845255	576	dbSNP
rs1157427491	587	dbSNP
rs1028663181	589	dbSNP
rs930056320	591	dbSNP
rs572205983	601	dbSNP
rs757976600	613	dbSNP
rs1451319216	615	dbSNP
rs1336760270	625	dbSNP
rs1384602795	629	dbSNP
rs1038605241	640	dbSNP
rs1258074092	641	dbSNP
rs1317276306	643	dbSNP
rs1240843058	648	dbSNP
rs1254212021	652	dbSNP
rs1460781721	667	dbSNP
rs1204058697	675	dbSNP
rs1464650159	680	dbSNP
rs1043996331	687	dbSNP
rs942540307	703	dbSNP
rs752232686	708	dbSNP
rs763666728	714	dbSNP
rs144773630	715	dbSNP
rs1210699512	718	dbSNP
rs1407457639	732	dbSNP
rs1420052824	740	dbSNP
rs1161680798	745	dbSNP
rs1056916825	747	dbSNP
rs1469718095	756	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.