>CTDNEP1|NM_001143775|3'UTR
   1 CAGCTGCTCCCCCTCCACCTGAGTTGGGGTGGGGGGGAAAGGGAGGGCGAGCCCTTGGGATGCCGTCTGATGCCCTGTCC
  81 AATGTGAGGACTGCCTGGGCAGGGTCTGCCCCTCCCACCCCTCTCTGCCCTGGGAGCCCTACACTCCACTTGGAGTCTGG
 161 ATGGACACATGGGCCAGGGGCTCTGAAGCAGCCTCACTCTTAACTTCGTGTTCACACTCCATGGAAACCCCAGACTGGGA
 241 CACAGGCGGAAGCCTAGGAGAGCCGAATCAGTGTTTGTGAAGAGGCAGGACTGGCCAGAGTGACAGACATACGGTGATCC
 321 AGGAGGCTCAAAGAGAAGCCAAGTCAGCTTTGTTGTGATTTGATTTTTTTTAAAAAACTCTTGTACAAAACTGATCTAAT
 401 TCTTCACTCCTGCTCCAAGGGCTGGGCTGTGGGTGGGATACTGGGATTTTGGGCCACTGGATTTTCCCTAAATTTGTCCC
 481 CCCTTTACTCTCCCTCTATTTTTCTCTCCTTAGACTCCCTCAGACCTGTAACCAGCTTTGTGTCTTTTTTCCTTTTCTCT
 561 CTTTTAAACCATGCATTATAACTTTGAAACCAAAGCGG

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cgucgGUCGA-GAUGGGGGCGGu 5' ||||| || ||||| || Target 5' -----CAGCTGCT-CCCCCTCCa 3'	1 - 17	132.00	-22.10
2	miRNA 3' cgUCGGUCGAGAUGGGGGCGGu 5' :|| :| |||: |||| || Target 5' tgGGCAGGGTCTG-CCCCTCCc 3'	96 - 116	127.00	-24.40
3	miRNA 3' cguCGGUCGAGAUGGGGGCGGu 5' |||: || |||:|:| Target 5' gatGCCGTCTGATGCCCTGTCc 3'	59 - 80	119.00	-17.80

Mutant ID	Mutant Position	Mutant Source
COSN26972304	9	COSMIC
COSN31493314	15	COSMIC
COSN5854002	30	COSMIC
COSN20054173	31	COSMIC
COSN31491907	54	COSMIC
COSN30542458	74	COSMIC
COSN19657103	151	COSMIC
COSN31490988	207	COSMIC
COSN5420409	207	COSMIC
COSN31515771	265	COSMIC
COSN30169796	313	COSMIC
COSN29315511	363	COSMIC
COSN9322225	478	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs749273308	2	dbSNP
rs1272377697	6	dbSNP
rs773104545	13	dbSNP
rs1326206686	23	dbSNP
rs769699264	24	dbSNP
rs747884394	25	dbSNP
rs780985493	30	dbSNP
rs751083423	31	dbSNP
rs779755424	31	dbSNP
rs1161136683	32	dbSNP
rs537063620	33	dbSNP
rs751035405	37	dbSNP
rs758395345	38	dbSNP
rs779366903	38	dbSNP
rs779546593	38	dbSNP
rs987228874	42	dbSNP
rs955460819	43	dbSNP
rs1474327866	46	dbSNP
rs757722724	47	dbSNP
rs754156391	48	dbSNP
rs373383465	49	dbSNP
rs1189531572	51	dbSNP
rs1025454289	58	dbSNP
rs1201536343	59	dbSNP
rs1388654448	63	dbSNP
rs767051566	64	dbSNP
rs757534410	65	dbSNP
rs1304202895	71	dbSNP
rs1013683808	75	dbSNP
rs1213633667	77	dbSNP
rs868635572	77	dbSNP
rs1359206366	82	dbSNP
rs1316565548	95	dbSNP
rs1291226235	99	dbSNP
rs375061566	103	dbSNP
rs1405986037	109	dbSNP
rs1022505153	121	dbSNP
rs1296760373	127	dbSNP
rs757142685	127	dbSNP
rs1012492151	128	dbSNP
rs895413415	130	dbSNP
rs1463795287	144	dbSNP
rs571313836	146	dbSNP
rs1320091927	149	dbSNP
rs34926505	154	dbSNP
rs397782425	154	dbSNP
rs397815162	154	dbSNP
rs1290398474	180	dbSNP
rs1055443410	183	dbSNP
rs371312829	191	dbSNP
rs938361829	196	dbSNP
rs1243876669	207	dbSNP
rs762347491	207	dbSNP
rs1188059998	208	dbSNP
rs906915803	208	dbSNP
rs1447039167	215	dbSNP
rs1184256792	216	dbSNP
rs1164544996	217	dbSNP
rs551507969	219	dbSNP
rs1046800297	220	dbSNP
rs929764675	221	dbSNP
rs1444161089	233	dbSNP
rs1043008610	235	dbSNP
rs1346651879	237	dbSNP
rs1435155266	240	dbSNP
rs1319376742	241	dbSNP
rs1362923140	243	dbSNP
rs534752723	247	dbSNP
rs575100595	248	dbSNP
rs973906013	248	dbSNP
rs1355415904	255	dbSNP
rs1286581568	258	dbSNP
rs1330157105	260	dbSNP
rs1201705029	263	dbSNP
rs1481455069	267	dbSNP
rs8930	269	dbSNP
rs1188894087	270	dbSNP
rs1251707148	271	dbSNP
rs1237216784	272	dbSNP
rs1472004276	274	dbSNP
rs565881349	280	dbSNP
rs942614215	283	dbSNP
rs945685685	286	dbSNP
rs1203322028	294	dbSNP
rs753337024	296	dbSNP
rs1305368288	301	dbSNP
rs1228894705	304	dbSNP
rs1385808251	308	dbSNP
rs1470891657	310	dbSNP
rs1334770099	312	dbSNP
rs911146302	312	dbSNP
rs1281130221	314	dbSNP
rs1051925532	317	dbSNP
rs1228022706	319	dbSNP
rs933067531	320	dbSNP
rs1325788669	322	dbSNP
rs776322228	323	dbSNP
rs986789403	327	dbSNP
rs922058056	331	dbSNP
rs1313467532	336	dbSNP
rs955560012	339	dbSNP
rs1441265069	356	dbSNP
rs974906995	361	dbSNP
rs1253000033	362	dbSNP
rs549190536	371	dbSNP
rs1181048112	372	dbSNP
rs529161062	372	dbSNP
rs909434086	372	dbSNP
rs983736448	372	dbSNP
rs978163004	373	dbSNP
rs968153551	377	dbSNP
rs1024983030	378	dbSNP
rs1370973635	380	dbSNP
rs1409391151	381	dbSNP
rs1286660457	385	dbSNP
rs1022455810	386	dbSNP
rs1347668602	389	dbSNP
rs569950308	396	dbSNP
rs1051449	400	dbSNP
rs992164013	407	dbSNP
rs1012441615	409	dbSNP
rs1367681102	413	dbSNP
rs895372793	414	dbSNP
rs1035691036	424	dbSNP
rs1002502621	426	dbSNP
rs1275741995	429	dbSNP
rs906859622	439	dbSNP
rs1482945660	441	dbSNP
rs768030065	444	dbSNP
rs1046747200	450	dbSNP
rs372251830	455	dbSNP
rs1249123739	456	dbSNP
rs1453039731	457	dbSNP
rs1198883075	466	dbSNP
rs1376579924	470	dbSNP
rs16956630	476	dbSNP
rs993863007	477	dbSNP
rs898273643	478	dbSNP
rs1164516563	479	dbSNP
rs16956628	482	dbSNP
rs1170439307	484	dbSNP
rs760186192	486	dbSNP
rs16956625	494	dbSNP
rs1383465631	495	dbSNP
rs1001311160	498	dbSNP
rs767548427	498	dbSNP
rs562519076	504	dbSNP
rs1387577173	509	dbSNP
rs904389168	511	dbSNP
rs775038886	518	dbSNP
rs1433440148	523	dbSNP
rs1298960027	524	dbSNP
rs1342124813	525	dbSNP
rs1010168127	526	dbSNP
rs1294420558	529	dbSNP
rs942539497	538	dbSNP
rs1210096369	544	dbSNP
rs1265999869	545	dbSNP
rs1189989394	551	dbSNP
rs1449357339	551	dbSNP
rs1185280065	552	dbSNP
rs1420831510	557	dbSNP
rs1251219700	559	dbSNP
rs1176404299	561	dbSNP
rs1458565508	563	dbSNP
rs1162910950	565	dbSNP
rs1388904241	570	dbSNP
rs1481982752	571	dbSNP
rs911106065	572	dbSNP
rs1270517842	580	dbSNP
rs549880804	583	dbSNP
rs933013815	590	dbSNP
rs1341469400	591	dbSNP
rs1244582454	595	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.