>ZNF467|NM_207336|3'UTR
   1 GCCTAGTTCTCACGAGGACCCTTTCTTGCCCACAGTTTCGAGAGGCCCGTGCCATGAGACCGCCTGGGGTGAGCAAGGCG
  81 ACCTGGGCTGCTGCCCGAAGGTTTGGCCGCCGCGGGACACCTGTTTCCTTCCCGCAGTGTCTGCGTCCGCACAGCATACC
 161 CAGCTCGGACCTCCTAGGACAGAGACTCAGCGAACCCTTGCTGGGAACCGCTGAGCTGAAGTTCTTGGAAGGCTCCCACC
 241 CAGGTGCCCCGTTGGAAAGCAGATATTTCCCGGACCCAGCGCGGCCTCAACCAGGGCAGGAAAGAGTGGTTATTTATGTA
 321 CTTAAAGTTTCATTAAAGTTAAAATCGGAAAGTTCTGGGGCTGCTAAATGAATTGGGGGAGGGAACACCTGACTCTCCTT
 401 AGCCACTGTCCCACCTCCATCCACACACAGACCACCCCCCTCCACTTCCCCTTCTGTCCTGGGTGAGTTACATTTAGCCA
 481 GCTGCTGTTAATTGGTTCTCCCAAATAAAAATGTAATATATTTATTCACTAAAAAAAAAAAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ugcaaauuUAAAGUGGGGAAGa 5' | || ||||||| Target 5' ccccctccACTT--CCCCTTCt 3'	436 - 455	142.00	-13.20
2	miRNA 3' ugcaaAUUUAAAG------UGGGGAAGa 5' ||: | || ||||:||| Target 5' --gccTAGTTCTCACGAGGACCCTTTCt 3'	1 - 26	131.00	-13.90
3	miRNA 3' ugcaaauuuaaaguGGGGAAGa 5' ::||||| Target 5' cgcgggacacctgtTTCCTTCc 3'	111 - 132	108.00	-9.50

Mutant ID	Mutant Position	Mutant Source
COSN30156405	11	COSMIC
COSN30180019	11	COSMIC
COSN23404300	19	COSMIC
COSN30635217	49	COSMIC
COSN29088127	80	COSMIC
COSN15643303	99	COSMIC
COSN14270224	114	COSMIC
COSN31531500	176	COSMIC
COSN30684533	191	COSMIC
COSN19773448	259	COSMIC
COSN31578887	280	COSMIC
COSN31482876	304	COSMIC
COSN14270218	346	COSMIC
COSN20076068	375	COSMIC
COSN30538750	411	COSMIC
COSN31521665	447	COSMIC
COSN8515909	450	COSMIC
COSN31536706	488	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs778080256	2	dbSNP
rs1469932161	5	dbSNP
rs1423551105	8	dbSNP
rs1422818719	13	dbSNP
rs772470092	14	dbSNP
rs1430054693	15	dbSNP
rs1043303937	16	dbSNP
rs1223339595	17	dbSNP
rs748532036	19	dbSNP
rs778959827	20	dbSNP
rs1187269319	21	dbSNP
rs1472592717	24	dbSNP
rs566991532	25	dbSNP
rs913364132	27	dbSNP
rs112370637	29	dbSNP
rs780241345	30	dbSNP
rs1352370032	31	dbSNP
rs1197334425	34	dbSNP
rs1263269379	38	dbSNP
rs757259688	39	dbSNP
rs933120832	40	dbSNP
rs199642677	42	dbSNP
rs984218732	44	dbSNP
rs1340774621	48	dbSNP
rs763959847	54	dbSNP
rs1452704860	61	dbSNP
rs758220150	62	dbSNP
rs752569759	65	dbSNP
rs764732782	66	dbSNP
rs1354829209	67	dbSNP
rs1163796031	69	dbSNP
rs962991759	70	dbSNP
rs188408041	71	dbSNP
rs868852101	75	dbSNP
rs765913456	77	dbSNP
rs1185700963	78	dbSNP
rs1445270706	80	dbSNP
rs1449918028	83	dbSNP
rs983354997	84	dbSNP
rs761144176	88	dbSNP
rs970911222	88	dbSNP
rs1481910039	91	dbSNP
rs1252477032	92	dbSNP
rs1369398496	96	dbSNP
rs773657017	97	dbSNP
rs10215169	99	dbSNP
rs1013828722	101	dbSNP
rs753103724	101	dbSNP
rs1246302940	106	dbSNP
rs748442360	107	dbSNP
rs1164605469	108	dbSNP
rs531754070	109	dbSNP
rs757641061	111	dbSNP
rs768749401	112	dbSNP
rs1292918625	113	dbSNP
rs749592141	114	dbSNP
rs1367245856	116	dbSNP
rs904493677	122	dbSNP
rs1286167273	123	dbSNP
rs1354421181	124	dbSNP
rs1425552425	127	dbSNP
rs1041506545	128	dbSNP
rs1379699993	128	dbSNP
rs1157009350	129	dbSNP
rs530972659	131	dbSNP
rs867153654	134	dbSNP
rs1258733780	138	dbSNP
rs1264664516	141	dbSNP
rs756315817	144	dbSNP
rs1320649356	145	dbSNP
rs374829113	146	dbSNP
rs945865185	148	dbSNP
rs946360841	153	dbSNP
rs1222680829	154	dbSNP
rs901030082	157	dbSNP
rs565268761	158	dbSNP
rs1472221637	161	dbSNP
rs1216612318	166	dbSNP
rs1258644323	171	dbSNP
rs942650589	171	dbSNP
rs1464036912	173	dbSNP
rs780861740	174	dbSNP
rs1317200066	175	dbSNP
rs1240531544	180	dbSNP
rs1317031318	187	dbSNP
rs751935539	190	dbSNP
rs1385027887	196	dbSNP
rs1432843629	201	dbSNP
rs1328216185	202	dbSNP
rs1347099481	203	dbSNP
rs758417097	209	dbSNP
rs855666	210	dbSNP
rs1342413857	211	dbSNP
rs755010191	219	dbSNP
rs765143039	222	dbSNP
rs984135587	226	dbSNP
rs921704022	234	dbSNP
rs931362572	235	dbSNP
rs918657347	236	dbSNP
rs1364494186	238	dbSNP
rs1328319982	245	dbSNP
rs778042450	246	dbSNP
rs1479743987	247	dbSNP
rs941708358	248	dbSNP
rs972843424	249	dbSNP
rs970914896	250	dbSNP
rs1488194037	251	dbSNP
rs1171930651	252	dbSNP
rs1259191963	253	dbSNP
rs753572195	259	dbSNP
rs1485880076	260	dbSNP
rs1254914893	261	dbSNP
rs1221541763	263	dbSNP
rs371444717	267	dbSNP
rs983518719	270	dbSNP
rs1170621832	272	dbSNP
rs950697175	280	dbSNP
rs375127100	283	dbSNP
rs199797137	297	dbSNP
rs185205901	299	dbSNP
rs73168053	316	dbSNP
rs1277572248	331	dbSNP
rs1332192829	375	dbSNP
rs73168052	375	dbSNP
rs968376416	377	dbSNP
rs765333996	379	dbSNP
rs866307391	383	dbSNP
rs968706885	398	dbSNP
rs117383860	404	dbSNP
rs1287710562	407	dbSNP
rs1487064599	411	dbSNP
rs1010641920	414	dbSNP
rs892073244	415	dbSNP
rs1009975262	417	dbSNP
rs1446606659	418	dbSNP
rs1434866051	419	dbSNP
rs1196630437	420	dbSNP
rs1353092772	423	dbSNP
rs1440065189	427	dbSNP
rs180763629	435	dbSNP
rs1041277743	436	dbSNP
rs1297684339	437	dbSNP
rs867417969	438	dbSNP
rs1006780224	439	dbSNP
rs1389083010	442	dbSNP
rs997708015	449	dbSNP
rs900644823	456	dbSNP
rs373244323	459	dbSNP
rs543901749	468	dbSNP
rs1311840536	495	dbSNP
rs889721012	516	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.