>CYP4A22|NM_001010969|3'UTR
   1 GGGCCTCCACCTGCCATCCTGTCTTCCTGACCCCCACTCCTATCTCCTGCCTGTCTG

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' aauucauuGUGGAAGGACUuau 5' :::|||||||| Target 5' tgccatccTGTCTTCCTGAccc 3'	12 - 33	118.00	-11.62
2	miRNA 3' aauucAUUGUGGAAGGACuuau 5' || | ||| |||| Target 5' actccTATCTCCTGCCTGtctg 3'	36 - 57	101.00	-9.06

Mutant ID	Mutant Position	Mutant Source
COSN6458434	17	COSMIC
COSN30145191	52	COSMIC
COSN15153936	153	COSMIC
COSN15168000	196	COSMIC
COSN6458435	264	COSMIC
COSN26144919	369	COSMIC
COSN16196923	524	COSMIC
COSN17940290	559	COSMIC
COSN17454749	676	COSMIC
COSN6036972	691	COSMIC
COSN16493731	904	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1383486681	3	dbSNP
rs751186069	5	dbSNP
rs756868067	10	dbSNP
rs200828882	11	dbSNP
rs750305500	14	dbSNP
rs375361646	16	dbSNP
rs72684327	17	dbSNP
rs1454046092	22	dbSNP
rs1306734583	24	dbSNP
rs1373130335	26	dbSNP
rs753558060	31	dbSNP
rs180918178	32	dbSNP
rs1386458638	34	dbSNP
rs1311660847	36	dbSNP
rs1208360409	37	dbSNP
rs1260237527	38	dbSNP
rs1458335905	43	dbSNP
rs1215192118	51	dbSNP
rs1485715925	54	dbSNP
rs550586068	60	dbSNP
rs568741433	70	dbSNP
rs1200727113	72	dbSNP
rs950453339	75	dbSNP
rs1271103930	80	dbSNP
rs1006026525	109	dbSNP
rs1295514714	111	dbSNP
rs1251578716	112	dbSNP
rs1016119672	113	dbSNP
rs1477899096	123	dbSNP
rs1192356791	131	dbSNP
rs1311195437	144	dbSNP
rs11211469	153	dbSNP
rs554308588	159	dbSNP
rs147169587	171	dbSNP
rs1489014569	186	dbSNP
rs1371203620	190	dbSNP
rs1169363244	194	dbSNP
rs10789502	196	dbSNP
rs964601631	201	dbSNP
rs1441439858	205	dbSNP
rs974710314	206	dbSNP
rs1323449811	213	dbSNP
rs1337857511	215	dbSNP
rs1257890767	217	dbSNP
rs923250080	224	dbSNP
rs1357963073	236	dbSNP
rs558953855	238	dbSNP
rs538589087	239	dbSNP
rs1336520074	245	dbSNP
rs558793577	247	dbSNP
rs954765286	248	dbSNP
rs1394052585	260	dbSNP
rs372793464	264	dbSNP
rs1419974381	267	dbSNP
rs544430072	273	dbSNP
rs1472184297	274	dbSNP
rs114643922	275	dbSNP
rs1220757069	276	dbSNP
rs1287993088	287	dbSNP
rs1476933753	295	dbSNP
rs1451189210	307	dbSNP
rs1170278859	310	dbSNP
rs1222785527	312	dbSNP
rs575153882	318	dbSNP
rs1485195000	320	dbSNP
rs1227747859	324	dbSNP
rs1351492393	329	dbSNP
rs1193532294	332	dbSNP
rs542452720	341	dbSNP
rs1440264858	349	dbSNP
rs1478507835	350	dbSNP
rs1330379344	357	dbSNP
rs1458235099	358	dbSNP
rs1396522475	359	dbSNP
rs184858551	360	dbSNP
rs528238739	361	dbSNP
rs1159842399	363	dbSNP
rs1405542958	367	dbSNP
rs539943441	369	dbSNP
rs1194596890	375	dbSNP
rs1343184676	376	dbSNP
rs1251597804	377	dbSNP
rs9333039	378	dbSNP
rs923987996	379	dbSNP
rs1222169788	380	dbSNP
rs531992184	390	dbSNP
rs1341281632	392	dbSNP
rs1401932686	393	dbSNP
rs1282446944	395	dbSNP
rs1355937041	396	dbSNP
rs936766111	406	dbSNP
rs1229060813	408	dbSNP
rs1292515369	409	dbSNP
rs1351929374	416	dbSNP
rs1398470554	418	dbSNP
rs779464865	420	dbSNP
rs149725375	421	dbSNP
rs1264525542	424	dbSNP
rs568827028	425	dbSNP
rs1191022579	432	dbSNP
rs189770568	434	dbSNP
rs9333040	441	dbSNP
rs868799500	446	dbSNP
rs1183217729	447	dbSNP
rs3176979	450	dbSNP
rs1213266191	455	dbSNP
rs1126748	456	dbSNP
rs1053852394	457	dbSNP
rs1277194056	459	dbSNP
rs1218454826	467	dbSNP
rs1362395254	471	dbSNP
rs1271866583	474	dbSNP
rs1432727759	482	dbSNP
rs1359335297	485	dbSNP
rs1332304631	491	dbSNP
rs1436163433	498	dbSNP
rs146771558	501	dbSNP
rs1429496832	510	dbSNP
rs1422186706	514	dbSNP
rs534340778	515	dbSNP
rs1126749	524	dbSNP
rs1258286122	538	dbSNP
rs1190198332	540	dbSNP
rs1375229313	542	dbSNP
rs552371632	543	dbSNP
rs1264170062	544	dbSNP
rs370563584	557	dbSNP
rs879336826	557	dbSNP
rs1126750	558	dbSNP
rs1126751	559	dbSNP
rs113510255	562	dbSNP
rs181715404	564	dbSNP
rs1293326766	575	dbSNP
rs1416111252	578	dbSNP
rs1376445010	579	dbSNP
rs867021414	584	dbSNP
rs113001906	585	dbSNP
rs1169043140	586	dbSNP
rs1465532363	591	dbSNP
rs536155798	592	dbSNP
rs1167127059	594	dbSNP
rs1474271005	595	dbSNP
rs1235986891	596	dbSNP
rs1438636843	597	dbSNP
rs1179050911	598	dbSNP
rs1381888987	601	dbSNP
rs1452544072	608	dbSNP
rs554450597	610	dbSNP
rs1401655572	614	dbSNP
rs572532066	630	dbSNP
rs1336173977	635	dbSNP
rs1446528694	640	dbSNP
rs186908559	641	dbSNP
rs1398392983	647	dbSNP
rs1456896227	650	dbSNP
rs1367944191	653	dbSNP
rs1159975936	655	dbSNP
rs540352569	656	dbSNP
rs1310499726	659	dbSNP
rs188994292	660	dbSNP
rs1379550228	666	dbSNP
rs1046679715	672	dbSNP
rs906861390	675	dbSNP
rs12725864	676	dbSNP
rs1264314091	682	dbSNP
rs1239173341	686	dbSNP
rs1318293719	687	dbSNP
rs576900734	688	dbSNP
rs1220470606	689	dbSNP
rs1372170731	701	dbSNP
rs1002506068	717	dbSNP
rs1047439105	725	dbSNP
rs1255551982	728	dbSNP
rs1457063049	729	dbSNP
rs1198846759	730	dbSNP
rs1239826677	736	dbSNP
rs886163094	744	dbSNP
rs1158268666	754	dbSNP
rs181788939	755	dbSNP
rs1454915003	756	dbSNP
rs1395427444	758	dbSNP
rs1449639815	761	dbSNP
rs12726029	762	dbSNP
rs1016085288	772	dbSNP
rs1378138412	774	dbSNP
rs1478935371	777	dbSNP
rs543813603	778	dbSNP
rs562096363	779	dbSNP
rs900360733	783	dbSNP
rs1306033386	784	dbSNP
rs1231560253	785	dbSNP
rs1330973752	789	dbSNP
rs996486123	795	dbSNP
rs1384488879	806	dbSNP
rs1030284668	813	dbSNP
rs138939887	820	dbSNP
rs1332284513	830	dbSNP
rs1174060071	832	dbSNP
rs978029342	836	dbSNP
rs1376926100	837	dbSNP
rs1193796843	840	dbSNP
rs1478792368	841	dbSNP
rs1030972580	845	dbSNP
rs1192341531	852	dbSNP
rs1402993453	856	dbSNP
rs1385461293	860	dbSNP
rs1280314809	861	dbSNP
rs113826273	865	dbSNP
rs1323243495	869	dbSNP
rs958091017	872	dbSNP
rs1332204351	875	dbSNP
rs989553654	878	dbSNP
rs1339141492	884	dbSNP
rs1277596565	889	dbSNP
rs1415496218	895	dbSNP
rs1357542840	901	dbSNP
rs374444842	904	dbSNP
rs548068403	907	dbSNP
rs1401212280	910	dbSNP
rs1168327370	920	dbSNP
rs1467084349	922	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.