>DUSP2|NM_004418|3'UTR
   1 GGTGGTGCCCCTCTGCCTGCCTGCCCCACTGTGCTGGCAGGAGCTGACTGTGGACTGGTGGGCTCCCCTCTGGGCCAGCA
  81 CAGTCCCCTCACCTCTGGCAGGGCTGCTACCTCCTCAGAGTTTCAGAAGCCCCCACATGGGGGCTCTAGGAATGCCGGCA
 161 TGCTGGTCTTTCCGACCTGGTGCTCTTCTGCTGGGGGACTGAGGCTGGCCCTCATTCGGGGTCGGGAACCAAGGGTGTGT
 241 CTGCTCTTTCCCTCCCCATCCTCTGGCAGAAATCAGCTAGACGCTATACCGTGGACTCTCCCTGGTCCACCACCATGTTG
 321 AAGCCCTTGGCAGCCTGAGAGCTCCAAGGAACAAGCTGTGACAACCAGGAGCCCTGTCTGTGGGTTCGTCTGCCCAGGGC
 401 CTGGAGCCCAAGCCCTGTGTTCCTGGGGAAGCTGGGGACTTGGGAAGTGATGGGTGTGTCATGTTGCGTGTGTCTGTCTG
 481 TGAGCCTTTCACACCTGTGCTGGCGCTGGAAAATTATTTGTGCTCAGCTGACATTTAACACTCCCTCCCCCGCTTCCTCC
 561 TAGCCCTGTGGGCAGGGGTTGGAAACTTAGCACTTTATATTTATACAGAACATTCAGGATATGTCAATAAAATATTGTTA
 641 TATTTAAAAAACAAAAAAAAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ggGUGG--UC-GACGAGGGACCGGGu 5' :||| :| || ||||||||:|| Target 5' taTACCGTGGACT-CTCCCTGGTCCa 3'	285 - 309	150.00	-30.00
2	miRNA 3' ggGUGGUCGAC-----GA--GGGACCGGGu 5' | :| |||| || ||||||| Target 5' ctCTTCTGCTGGGGGACTGAGGCTGGCCCt 3'	183 - 212	143.00	-19.70
3	miRNA 3' ggGUGGUCGACGAGGGACCGGGu 5' | || ||| | :||| |||| Target 5' ctCCCCCGCTTCCTCCTAGCCCt 3'	545 - 567	137.00	-24.20

Mutant ID	Mutant Position	Mutant Source
COSN31511196	7	COSMIC
COSN30526310	13	COSMIC
COSN30109085	23	COSMIC
COSN30465236	25	COSMIC
COSN31556466	28	COSMIC
COSN30115715	67	COSMIC
COSN30451923	75	COSMIC
COSN24299415	106	COSMIC
COSN31600943	116	COSMIC
COSN31541357	541	COSMIC
COSN31534858	630	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1225175504	2	dbSNP
rs747120101	5	dbSNP
rs1323184104	6	dbSNP
rs777787330	7	dbSNP
rs758609462	9	dbSNP
rs752968740	11	dbSNP
rs146609611	19	dbSNP
rs755931212	22	dbSNP
rs750307086	25	dbSNP
rs1478585039	26	dbSNP
rs767499624	28	dbSNP
rs761159833	30	dbSNP
rs374262102	31	dbSNP
rs1263138822	34	dbSNP
rs1201190548	53	dbSNP
rs1429530937	57	dbSNP
rs1192114042	59	dbSNP
rs996559056	61	dbSNP
rs1173675756	63	dbSNP
rs1397930024	70	dbSNP
rs1410476498	84	dbSNP
rs1489786671	85	dbSNP
rs900963641	86	dbSNP
rs1264195771	91	dbSNP
rs558374852	93	dbSNP
rs1333805095	103	dbSNP
rs1019319179	105	dbSNP
rs1438320685	113	dbSNP
rs115523494	116	dbSNP
rs111226214	122	dbSNP
rs1346418947	132	dbSNP
rs1223060519	147	dbSNP
rs746214027	156	dbSNP
rs780776323	157	dbSNP
rs995397150	165	dbSNP
rs749520869	166	dbSNP
rs1042361	173	dbSNP
rs923720004	174	dbSNP
rs977824362	179	dbSNP
rs1196763530	180	dbSNP
rs1055166250	192	dbSNP
rs937993341	195	dbSNP
rs1475437882	196	dbSNP
rs917444520	203	dbSNP
rs993378151	210	dbSNP
rs1426427815	214	dbSNP
rs958996869	217	dbSNP
rs111938402	218	dbSNP
rs1046506416	223	dbSNP
rs1303941300	224	dbSNP
rs1328323000	230	dbSNP
rs1724120	231	dbSNP
rs777355822	236	dbSNP
rs1025711413	242	dbSNP
rs1248725045	246	dbSNP
rs994191649	250	dbSNP
rs1292875415	254	dbSNP
rs1490284994	256	dbSNP
rs990139719	260	dbSNP
rs958278539	266	dbSNP
rs921506603	282	dbSNP
rs375931869	283	dbSNP
rs758445503	287	dbSNP
rs1019455834	290	dbSNP
rs1170527900	291	dbSNP
rs1446381443	304	dbSNP
rs1428112422	307	dbSNP
rs975087448	310	dbSNP
rs1384447999	317	dbSNP
rs781767087	319	dbSNP
rs1292056352	323	dbSNP
rs1387449736	327	dbSNP
rs889712263	334	dbSNP
rs371301983	341	dbSNP
rs570047731	342	dbSNP
rs1297999199	347	dbSNP
rs144243944	355	dbSNP
rs1363052805	359	dbSNP
rs551067232	365	dbSNP
rs1000563304	369	dbSNP
rs1316347504	382	dbSNP
rs1359234261	383	dbSNP
rs1253398709	388	dbSNP
rs539952138	393	dbSNP
rs902163000	399	dbSNP
rs752636140	407	dbSNP
rs371306999	412	dbSNP
rs183938631	419	dbSNP
rs1204123173	423	dbSNP
rs1259122893	430	dbSNP
rs765201877	431	dbSNP
rs1202381934	434	dbSNP
rs1462263813	438	dbSNP
rs1184173688	456	dbSNP
rs766031227	458	dbSNP
rs1256222129	459	dbSNP
rs1234883013	460	dbSNP
rs1003894730	467	dbSNP
rs759287617	468	dbSNP
rs1364095946	471	dbSNP
rs949130109	472	dbSNP
rs1158199278	474	dbSNP
rs570855741	476	dbSNP
rs1386633615	482	dbSNP
rs1470979286	483	dbSNP
rs1310235319	494	dbSNP
rs917624864	496	dbSNP
rs547654715	503	dbSNP
rs1026787084	504	dbSNP
rs1382738210	505	dbSNP
rs192294242	509	dbSNP
rs1057304846	512	dbSNP
rs777794683	515	dbSNP
rs1265810858	521	dbSNP
rs894433467	528	dbSNP
rs1055703561	544	dbSNP
rs115786506	547	dbSNP
rs906506700	551	dbSNP
rs1046533004	552	dbSNP
rs945440160	561	dbSNP
rs57072789	564	dbSNP
rs529185172	565	dbSNP
rs1408072429	568	dbSNP
rs972678584	571	dbSNP
rs1410060942	582	dbSNP
rs1481517647	586	dbSNP
rs531488809	587	dbSNP
rs1177252002	597	dbSNP
rs1369952012	597	dbSNP
rs1303656787	608	dbSNP
rs1053989264	620	dbSNP
rs1300408694	622	dbSNP
rs1397706181	623	dbSNP
rs936831287	626	dbSNP
rs1020077093	632	dbSNP
rs1286797017	634	dbSNP
rs1347045418	643	dbSNP
rs921552993	644	dbSNP
rs561811658	650	dbSNP
rs752112607	652	dbSNP
rs1251787907	655	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.