>ALOX15|NM_001140|3'UTR
   1 GCGTCGCCACCCTTTGGTTATTTCAGCCCCCATCACCCAAGCCACAAGCTGACCCCTTCGTGGTTATAGCCCTGCCCTCC
  81 CAAGTCCCACCCTCTTCCCATGTCCCACCCTCCCTAGAGGGGCACCTTTTCATGGTCTCTGCACCCAGTGAACACATTTT
 161 ACTCTAGAGGCATCACCTGGGACCTTACTCCTCTTTCCTTCCTTCCTCCTTTCCTATCTTCCTTCCTCTCTCTCTTCCTC
 241 TTTCTTCATTCAGATCTATATGGCAAATAGCCACAATTATATAAATCATTTCAAGACTAGAATAGGGGGATATAATACAT
 321 ATTACTCCACACCTTTTATGAATCAAATATGATTTTTTTGTTGTTGTTAAGACAGAGTCTCACTTTGACACCCAGGCTGG
 401 AGTGCAGTGGTGCCATCACCACGGCTCACTGCAGCCTCAGCGTCCTGGGCTCAAATGATCCTCCCACCTCAGCCTCCTGA
 481 GTAGCTGGGACTACAGGCTCATGCCATCATGCCCAGCTAATATTTTTTTATTTTCGTGGAGACGGGGCCTCACTATGTTG
 561 CCTAGGCTGGAAATAGGATTTTGAACCCAAATTGAGTTTAACAATAATAAAAAGTTGTTTTACGCTAAAGATGGAAAAGA
 641 ACTAGGACTGAACTATTTTAAATAAAATATTGGCAAAAGAAAAAAAAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' guaccAGGGUGGAGGGACGGAc 5' |||:|:||:||| ||| Target 5' tccttTCCTATCTTCCTTCCTc 3'	207 - 228	141.00	-20.50
2	miRNA 3' guACCAGGGUGGAGGGACGGac 5' |||| :|: ||||||| Target 5' cgTGGT--TATAGCCCTGCCct 3'	59 - 78	136.00	-21.70
3	miRNA 3' guaccAGGGU----GGAGGGACGGAc 5' ||||| || |||| ||| Target 5' cctctTCCCATGTCCCACCCTCCCTa 3'	91 - 116	131.00	-24.12

Mutant ID	Mutant Position	Mutant Source
COSN31606031	6	COSMIC
COSN31586045	49	COSMIC
COSN6657497	61	COSMIC
COSN30190033	62	COSMIC
COSN30464066	80	COSMIC
COSN31587032	87	COSMIC
COSN31560762	153	COSMIC
COSN28212525	507	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs976133157	2	dbSNP
rs149225580	3	dbSNP
rs746160458	5	dbSNP
rs367886148	6	dbSNP
rs1334112696	11	dbSNP
rs371452789	12	dbSNP
rs1178855174	22	dbSNP
rs1349452458	23	dbSNP
rs1413206549	25	dbSNP
rs777453762	27	dbSNP
rs755568166	28	dbSNP
rs989156282	30	dbSNP
rs752320206	31	dbSNP
rs1430902251	32	dbSNP
rs767291618	33	dbSNP
rs759534675	35	dbSNP
rs768651153	36	dbSNP
rs751354299	38	dbSNP
rs747060783	39	dbSNP
rs762887378	45	dbSNP
rs1456449634	47	dbSNP
rs768986002	49	dbSNP
rs1351645557	52	dbSNP
rs1223217280	59	dbSNP
rs905495926	60	dbSNP
rs916055	61	dbSNP
rs1272625797	62	dbSNP
rs1344638894	63	dbSNP
rs1174535174	67	dbSNP
rs1013958289	69	dbSNP
rs909827249	72	dbSNP
rs1252366137	76	dbSNP
rs371838334	80	dbSNP
rs951324992	85	dbSNP
rs1270778715	89	dbSNP
rs1478258726	93	dbSNP
rs1455697869	99	dbSNP
rs1036519685	100	dbSNP
rs1005433143	109	dbSNP
rs760691070	110	dbSNP
rs1404867150	112	dbSNP
rs1040815472	113	dbSNP
rs888055680	113	dbSNP
rs373560921	114	dbSNP
rs1437654959	122	dbSNP
rs1269783623	125	dbSNP
rs1429698654	126	dbSNP
rs1216345937	131	dbSNP
rs752811780	141	dbSNP
rs544767313	142	dbSNP
rs1264440059	144	dbSNP
rs1275428011	153	dbSNP
rs1358898896	155	dbSNP
rs958665138	156	dbSNP
rs931979353	158	dbSNP
rs921919091	170	dbSNP
rs1040518993	172	dbSNP
rs376040516	174	dbSNP
rs575821607	180	dbSNP
rs1293929352	183	dbSNP
rs1451921897	187	dbSNP
rs944752858	195	dbSNP
rs1239943935	205	dbSNP
rs1476180879	208	dbSNP
rs978779583	209	dbSNP
rs1280366777	211	dbSNP
rs968954727	213	dbSNP
rs1212584274	214	dbSNP
rs1020987586	223	dbSNP
rs1422265887	225	dbSNP
rs916056	225	dbSNP
rs1010367571	226	dbSNP
rs1393553191	230	dbSNP
rs913083815	234	dbSNP
rs12051603	236	dbSNP
rs775526353	258	dbSNP
rs1328167376	260	dbSNP
rs957408809	266	dbSNP
rs1298664445	277	dbSNP
rs925894036	280	dbSNP
rs1406311461	281	dbSNP
rs1339948727	284	dbSNP
rs11568131	287	dbSNP
rs1317028247	304	dbSNP
rs1436880969	305	dbSNP
rs997905407	305	dbSNP
rs1204544097	306	dbSNP
rs1281875254	307	dbSNP
rs969736851	309	dbSNP
rs11568132	310	dbSNP
rs796290152	311	dbSNP
rs1172400589	312	dbSNP
rs1039327740	346	dbSNP
rs1442196002	351	dbSNP
rs1370647415	360	dbSNP
rs1473406045	360	dbSNP
rs149949350	360	dbSNP
rs941071022	360	dbSNP
rs539035649	363	dbSNP
rs201563536	368	dbSNP
rs200868987	369	dbSNP
rs369225171	369	dbSNP
rs1168990763	375	dbSNP
rs1459203381	377	dbSNP
rs145398728	382	dbSNP
rs1321413841	389	dbSNP
rs1428372181	396	dbSNP
rs759165158	401	dbSNP
rs1023907609	404	dbSNP
rs1189426706	416	dbSNP
rs1484522501	419	dbSNP
rs1013988139	420	dbSNP
rs1277750593	421	dbSNP
rs561559450	422	dbSNP
rs553393338	423	dbSNP
rs770548157	426	dbSNP
rs1459710168	427	dbSNP
rs1205243358	428	dbSNP
rs1005056285	441	dbSNP
rs1436763391	445	dbSNP
rs1249340258	449	dbSNP
rs1181697905	450	dbSNP
rs927399076	454	dbSNP
rs78305230	456	dbSNP
rs978748375	463	dbSNP
rs866056696	465	dbSNP
rs1429095230	479	dbSNP
rs1468424735	485	dbSNP
rs1304847106	488	dbSNP
rs1402968560	490	dbSNP
rs968711693	492	dbSNP
rs184061120	500	dbSNP
rs1410140909	506	dbSNP
rs989488174	509	dbSNP
rs1416668752	511	dbSNP
rs1429299830	513	dbSNP
rs1049376014	516	dbSNP
rs778006671	520	dbSNP
rs900525722	522	dbSNP
rs1353855788	523	dbSNP
rs1193609030	524	dbSNP
rs1030905668	530	dbSNP
rs954873482	530	dbSNP
rs1040382855	535	dbSNP
rs568323228	536	dbSNP
rs11568134	538	dbSNP
rs1296753063	540	dbSNP
rs1378458775	541	dbSNP
rs538005194	543	dbSNP
rs1166242736	549	dbSNP
rs568615564	555	dbSNP
rs375797237	559	dbSNP
rs925779782	560	dbSNP
rs191843826	561	dbSNP
rs1416476201	562	dbSNP
rs969936520	590	dbSNP
rs929868247	591	dbSNP
rs895756267	599	dbSNP
rs1224542511	605	dbSNP
rs1276145909	609	dbSNP
rs1182090950	618	dbSNP
rs1342301937	620	dbSNP
rs1056982794	622	dbSNP
rs917135621	623	dbSNP
rs992472164	624	dbSNP
rs1268244794	625	dbSNP
rs1428127694	627	dbSNP
rs532070814	630	dbSNP
rs537461917	633	dbSNP
rs1162800202	639	dbSNP
rs913239451	640	dbSNP
rs1274949990	641	dbSNP
rs1459193635	642	dbSNP
rs1293912789	653	dbSNP
rs1384052053	660	dbSNP
rs1005707960	669	dbSNP
rs1380134956	675	dbSNP
rs546555515	678	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.