>PPP1R11|NM_021959|3'UTR
   1 ATCCCTCTCTCCTCCAGCATTCCTGTGTCTGTCTGGCCCTAAATGTATCCATGTGGCTACTTCTCCAGCCCCCTCCTTCC
  81 CTCTCTTCTGCCTGATAGAGGGAAGAGGAAGAGGAGGACGAACAGAGATCCTGAAATTCTGACTTGCTGCTATTCCAGAA
 161 CCCAGCCTCCTGGGTTTCCCCAGTCCTCATTTTTCCTCCCAATACCCACCCTTCTCTCTCGAGGGATCTAGGCACCTTGG
 241 TCCCAGTGTCTTCCTTTTGTTCTCACTGCCAAACTGCCTGTCCTGGGATCTAGTTATCTTGGCCCTGCACTCTCAACATG
 321 AGTAGCGAACACTTAAATTGGGTTTTCAACAGTCCCAGCTTTCACTGCCAGGGTCCCAGTCAGATTCCAGGAATTTGCGC
 401 CCTAACTTTGCTTGCTAATCCTGGTTTAGAGCTATCCCACTAAAATATTTAATCCTAATTCTTAGTCCTTGCCTGTGAGA
 481 TATGAGGTCTTACAGGAGACCTCAGAGCTCCCAGCCCTTCTCCTCCTGCTAACCCTTCTCACACCCTCAAGAGGAGTTAG
 561 AAAAGAGGTCCTTGTCATTCTCACCTCTTATGGAAAATGGAATAAGAAATAATCATATCCTTTCTTCCCACCCTTCTCCT
 641 GTTATTTAGGATTTCTGACAAAGCTGGCTTGAGATTGGTCACTTAGAGCCGACTGTCTCCTCTGCCTTTTGTTTTTCAGC
 721 TTCAGAGACAGATCCAATATAGTCCCAGGGACCTGGGTCTCTGGGAGAGGAAGGAAGAGGGAGGGAGCAAAGAGATTGGG
 801 GTATGTCCCCTGTAGTACACTCTTACCTCTTACTTCCTAGACTTTGATTTCTCCGGCAGCCCAGATGTTCAGTTCTCTTG
 881 GCCCCTCTCTACCCCTTACTGGGATCCGGTTTTCATTTTCCGGTCCTTTTGCCATACACAGTTACAGAGATCAGTCAAAT
 961 CCATACCACCACTGAGATCTCATTTATTGCCACAGATGCACAAAATAAATAACCCAAAATCACAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uucacccGACCCUAGGCCCGAc 5' ||||||||||| :| Target 5' ccccttaCTGGGATCCGGTTTt 3'	892 - 913	127.00	-22.90
2	miRNA 3' uucacccGACCCUAGGCCC-GAc 5' | |||| |:||| || Target 5' atagtccCAGGGACCTGGGTCTc 3'	739 - 761	111.00	-15.00
3	miRNA 3' uucacccgacccuaGGCCCGAc 5' |:|||:| Target 5' cagaacccagcctcCTGGGTTt 3'	156 - 177	108.00	-10.90

Mutant ID	Mutant Position	Mutant Source
COSN30159376	4	COSMIC
COSN30478804	4	COSMIC
COSN30486764	5	COSMIC
COSN31599999	65	COSMIC
COSN30172918	66	COSMIC
COSN30495389	94	COSMIC
COSN30157304	99	COSMIC
COSN31548225	130	COSMIC
COSN31562048	151	COSMIC
COSN30108732	221	COSMIC
COSN28695733	271	COSMIC
COSN21017766	328	COSMIC
COSN31962605	364	COSMIC
COSN31513265	373	COSMIC
COSN27055675	594	COSMIC
COSN28693749	975	COSMIC
rs6539	908	GWAS

Mutant ID	Mutant Position	Mutant Source
rs1199116754	3	dbSNP
rs1490633555	8	dbSNP
rs1268474344	14	dbSNP
rs1031339343	15	dbSNP
rs1179183273	23	dbSNP
rs767896119	27	dbSNP
rs753664615	32	dbSNP
rs1162865778	40	dbSNP
rs540656192	41	dbSNP
rs370011121	47	dbSNP
rs1353595762	54	dbSNP
rs1271714373	66	dbSNP
rs1219112104	67	dbSNP
rs1439536233	69	dbSNP
rs1319884051	70	dbSNP
rs185477475	71	dbSNP
rs914479779	74	dbSNP
rs1295439918	82	dbSNP
rs532503020	102	dbSNP
rs953906616	115	dbSNP
rs371455524	120	dbSNP
rs910660063	121	dbSNP
rs1467288753	130	dbSNP
rs375944037	135	dbSNP
rs1344155356	144	dbSNP
rs1480933236	163	dbSNP
rs763335445	168	dbSNP
rs569339833	173	dbSNP
rs1192528763	176	dbSNP
rs1040820262	195	dbSNP
rs570172216	203	dbSNP
rs531558835	221	dbSNP
rs919282238	221	dbSNP
rs369235796	222	dbSNP
rs768570003	225	dbSNP
rs1228895343	226	dbSNP
rs929262983	231	dbSNP
rs1049369328	241	dbSNP
rs1437502100	245	dbSNP
rs1221913204	247	dbSNP
rs1372838993	255	dbSNP
rs9261288	271	dbSNP
rs1449067175	280	dbSNP
rs1239343197	286	dbSNP
rs1013124695	297	dbSNP
rs1336568250	316	dbSNP
rs1470605405	316	dbSNP
rs568380079	318	dbSNP
rs1361210469	323	dbSNP
rs533838544	327	dbSNP
rs189714956	328	dbSNP
rs1406784985	342	dbSNP
rs1178880829	348	dbSNP
rs528568418	350	dbSNP
rs1044578138	374	dbSNP
rs1168899379	378	dbSNP
rs1419228980	393	dbSNP
rs907324509	395	dbSNP
rs1213384421	398	dbSNP
rs751706395	399	dbSNP
rs180960420	400	dbSNP
rs955732905	410	dbSNP
rs1380621038	412	dbSNP
rs1011176502	432	dbSNP
rs1396724754	437	dbSNP
rs1381931131	458	dbSNP
rs1287699558	484	dbSNP
rs1346163340	486	dbSNP
rs1335777993	499	dbSNP
rs1359311713	509	dbSNP
rs1158837106	520	dbSNP
rs9261289	534	dbSNP
rs1383055091	539	dbSNP
rs1334061700	541	dbSNP
rs953958995	544	dbSNP
rs1423959620	545	dbSNP
rs1258170931	550	dbSNP
rs1184718190	552	dbSNP
rs373487322	557	dbSNP
rs11543131	559	dbSNP
rs781601379	575	dbSNP
rs2240067	577	dbSNP
rs552227100	592	dbSNP
rs8347	604	dbSNP
rs1245606319	620	dbSNP
rs1323642546	620	dbSNP
rs1293975507	625	dbSNP
rs528454228	627	dbSNP
rs1341467534	633	dbSNP
rs115882910	635	dbSNP
rs554847473	637	dbSNP
rs115855070	644	dbSNP
rs1292648187	658	dbSNP
rs1049089458	666	dbSNP
rs1369697171	667	dbSNP
rs909178519	668	dbSNP
rs1424305557	672	dbSNP
rs1195533955	673	dbSNP
rs1241327016	682	dbSNP
rs1466678748	686	dbSNP
rs948951822	691	dbSNP
rs374353348	692	dbSNP
rs1210666750	695	dbSNP
rs1489483170	730	dbSNP
rs1249711986	731	dbSNP
rs560727348	738	dbSNP
rs377737924	743	dbSNP
rs1319170199	748	dbSNP
rs1280719328	750	dbSNP
rs1474635154	751	dbSNP
rs1222049655	754	dbSNP
rs1345284131	764	dbSNP
rs1300090186	770	dbSNP
rs1387029031	780	dbSNP
rs1392201313	781	dbSNP
rs771984764	797	dbSNP
rs116237627	798	dbSNP
rs1464686547	802	dbSNP
rs1052894912	806	dbSNP
rs1429584902	818	dbSNP
rs1468928645	822	dbSNP
rs1051133	826	dbSNP
rs1011233692	827	dbSNP
rs1021256568	837	dbSNP
rs1435104552	838	dbSNP
rs953676517	855	dbSNP
rs1195295144	856	dbSNP
rs1006829034	883	dbSNP
rs186201160	886	dbSNP
rs1202337357	895	dbSNP
rs1323232550	897	dbSNP
rs1256978845	900	dbSNP
rs6539	908	dbSNP
rs768114414	909	dbSNP
rs1304504763	917	dbSNP
rs1438357372	921	dbSNP
rs1445388150	922	dbSNP
rs776368761	935	dbSNP
rs554336785	938	dbSNP
rs770230037	940	dbSNP
rs1468187948	948	dbSNP
rs1346435639	950	dbSNP
rs950786314	951	dbSNP
rs1467451160	961	dbSNP
rs551000157	962	dbSNP
rs144268715	970	dbSNP
rs12526	975	dbSNP
rs1205723308	983	dbSNP
rs1351943516	983	dbSNP
rs948670342	987	dbSNP
rs1240143468	1017	dbSNP
rs1182236160	1019	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.