ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uacAUCCGGAAAUCUAGUGAa 5' || :: |||||| ||| Target 5' gaaTACATTGTTAGATGACTg 3'	760 - 780	126.00	-7.10
2	miRNA 3' uacauccggaaaucUAGUGAa 5' |||||| Target 5' ttattaaaatgcttATCACTg 3'	117 - 137	120.00	-5.50
3	miRNA 3' uacauccggaaaucUAGUGAa 5' |||||| Target 5' cattgtgtggatgtATCACTa 3'	1031 - 1051	120.00	-7.90

Mutant ID	Mutant Position	Mutant Source
COSN31575368	31	COSMIC
COSN31601593	113	COSMIC
COSN15661562	380	COSMIC
COSN27153221	393	COSMIC
COSN31959500	520	COSMIC
COSN20090460	714	COSMIC
COSN23742194	903	COSMIC
COSN22588480	954	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1457372761	3	dbSNP
rs1471445883	4	dbSNP
rs375499871	6	dbSNP
rs746487651	7	dbSNP
rs1037791096	8	dbSNP
rs770592621	10	dbSNP
rs776306471	11	dbSNP
rs759185943	15	dbSNP
rs1477646324	17	dbSNP
rs1489384940	17	dbSNP
rs769069105	30	dbSNP
rs897647056	31	dbSNP
rs1299522590	34	dbSNP
rs1165910687	36	dbSNP
rs1318812543	39	dbSNP
rs1229155432	41	dbSNP
rs774721615	43	dbSNP
rs1327033398	45	dbSNP
rs993312122	52	dbSNP
rs1230458389	75	dbSNP
rs1327412490	99	dbSNP
rs993590444	105	dbSNP
rs1208857246	110	dbSNP
rs1405936397	110	dbSNP
rs1242008522	111	dbSNP
rs1459653258	114	dbSNP
rs531376399	118	dbSNP
rs547861220	132	dbSNP
rs1189191912	134	dbSNP
rs1239573293	136	dbSNP
rs759587794	137	dbSNP
rs1345269539	138	dbSNP
rs906426049	142	dbSNP
rs1455267105	144	dbSNP
rs1410779209	158	dbSNP
rs1158473845	163	dbSNP
rs1002135736	166	dbSNP
rs1434711997	168	dbSNP
rs887720627	171	dbSNP
rs1006035054	172	dbSNP
rs1033552573	179	dbSNP
rs958772030	184	dbSNP
rs1033326683	186	dbSNP
rs539274563	192	dbSNP
rs1405459773	193	dbSNP
rs1234354865	200	dbSNP
rs1314280895	204	dbSNP
rs1299728816	206	dbSNP
rs1219146655	208	dbSNP
rs191570404	226	dbSNP
rs533856824	227	dbSNP
rs1340833336	230	dbSNP
rs989220313	232	dbSNP
rs1432300906	233	dbSNP
rs1336954302	234	dbSNP
rs1387393639	234	dbSNP
rs1020667167	236	dbSNP
rs558626165	243	dbSNP
rs1331066293	248	dbSNP
rs1175164084	252	dbSNP
rs7552305	253	dbSNP
rs1428987650	259	dbSNP
rs1228939721	261	dbSNP
rs1391951909	271	dbSNP
rs1190544368	275	dbSNP
rs572067465	276	dbSNP
rs977853642	287	dbSNP
rs1484394931	300	dbSNP
rs1254576165	302	dbSNP
rs547249245	303	dbSNP
rs1209862949	311	dbSNP
rs1201804162	316	dbSNP
rs966863387	317	dbSNP
rs977591226	318	dbSNP
rs1226905906	330	dbSNP
rs1363787962	335	dbSNP
rs570315953	342	dbSNP
rs923444504	343	dbSNP
rs1315221165	365	dbSNP
rs1397402410	368	dbSNP
rs954947656	384	dbSNP
rs986372551	385	dbSNP
rs910551868	393	dbSNP
rs114923651	394	dbSNP
rs1462332361	401	dbSNP
rs1372273410	408	dbSNP
rs1166612052	409	dbSNP
rs1429107598	411	dbSNP
rs1389621391	415	dbSNP
rs990268407	426	dbSNP
rs559049493	438	dbSNP
rs1037674530	439	dbSNP
rs919310288	442	dbSNP
rs1250094890	460	dbSNP
rs1473825098	461	dbSNP
rs929400457	470	dbSNP
rs1046201322	475	dbSNP
rs1428680491	476	dbSNP
rs1265455080	480	dbSNP
rs1464662504	488	dbSNP
rs140243321	491	dbSNP
rs1040078231	501	dbSNP
rs901616113	502	dbSNP
rs1174515655	505	dbSNP
rs928976196	507	dbSNP
rs1409645080	517	dbSNP
rs1409079451	521	dbSNP
rs1305887909	523	dbSNP
rs35345463	523	dbSNP
rs1002119403	526	dbSNP
rs1055080471	527	dbSNP
rs1047874118	530	dbSNP
rs1366844067	539	dbSNP
rs538268272	541	dbSNP
rs775917494	544	dbSNP
rs1006587755	548	dbSNP
rs1180484813	555	dbSNP
rs1482976135	561	dbSNP
rs1021056447	563	dbSNP
rs1211056461	566	dbSNP
rs1353613845	567	dbSNP
rs1264389671	574	dbSNP
rs966454150	578	dbSNP
rs894955980	581	dbSNP
rs1330792223	587	dbSNP
rs1282624199	589	dbSNP
rs554690082	593	dbSNP
rs1409023586	596	dbSNP
rs1245438559	600	dbSNP
rs1331562802	604	dbSNP
rs1030482582	607	dbSNP
rs1024382525	609	dbSNP
rs971911401	612	dbSNP
rs1325375250	616	dbSNP
rs1256108146	619	dbSNP
rs954916156	622	dbSNP
rs574853202	630	dbSNP
rs1472479914	645	dbSNP
rs1272851136	655	dbSNP
rs977444852	657	dbSNP
rs1032121755	660	dbSNP
rs957400323	663	dbSNP
rs990324352	676	dbSNP
rs748163587	681	dbSNP
rs910824849	685	dbSNP
rs540934849	689	dbSNP
rs1345416222	691	dbSNP
rs202111790	694	dbSNP
rs1230957150	707	dbSNP
rs765516089	709	dbSNP
rs750716115	710	dbSNP
rs973405888	712	dbSNP
rs1047385076	714	dbSNP
rs560717365	714	dbSNP
rs577559002	717	dbSNP
rs1403347367	719	dbSNP
rs941722689	720	dbSNP
rs554688456	723	dbSNP
rs1430220768	732	dbSNP
rs1046956230	734	dbSNP
rs927810962	739	dbSNP
rs546407339	743	dbSNP
rs937792122	749	dbSNP
rs1388437727	754	dbSNP
rs1388469978	759	dbSNP
rs537617894	762	dbSNP
rs1054966013	765	dbSNP
rs1055019493	781	dbSNP
rs895091746	798	dbSNP
rs1301288907	800	dbSNP
rs1013406671	811	dbSNP
rs1280402683	818	dbSNP
rs562636860	829	dbSNP
rs780235462	831	dbSNP
rs531283211	836	dbSNP
rs907275608	841	dbSNP
rs755445983	844	dbSNP
rs1031757993	851	dbSNP
rs182165783	855	dbSNP
rs1341305611	860	dbSNP
rs1430211501	860	dbSNP
rs1336517139	885	dbSNP
rs1344730688	885	dbSNP
rs1229660351	888	dbSNP
rs1375276423	888	dbSNP
rs561545487	894	dbSNP
rs1011547510	901	dbSNP
rs867234842	902	dbSNP
rs1185442961	903	dbSNP
rs1472882897	905	dbSNP
rs902202922	907	dbSNP
rs549867020	911	dbSNP
rs1275131742	912	dbSNP
rs1055642	916	dbSNP
rs1268137371	926	dbSNP
rs1464544085	935	dbSNP
rs1340035947	936	dbSNP
rs1186883341	937	dbSNP
rs976218895	940	dbSNP
rs1357264583	943	dbSNP
rs1392298158	949	dbSNP
rs1446142354	950	dbSNP
rs1029861739	955	dbSNP
rs1309082901	960	dbSNP
rs1412157141	962	dbSNP
rs1369248252	963	dbSNP
rs1450358072	967	dbSNP
rs1163748357	969	dbSNP
rs953807082	971	dbSNP
rs1459319569	978	dbSNP
rs748458801	988	dbSNP
rs1474713467	989	dbSNP
rs770171680	991	dbSNP
rs1383604424	997	dbSNP
rs963568223	999	dbSNP
rs1251024308	1002	dbSNP
rs1322105492	1007	dbSNP
rs150347670	1025	dbSNP
rs974093501	1029	dbSNP
rs1292200568	1038	dbSNP
rs1026310813	1045	dbSNP
rs138145041	1048	dbSNP
rs982140920	1052	dbSNP
rs1289302438	1057	dbSNP
rs949315250	1062	dbSNP
rs1298083606	1064	dbSNP
rs570405335	1068	dbSNP
rs532995891	1079	dbSNP
rs1321075165	1087	dbSNP
rs777370555	1088	dbSNP
rs748986135	1093	dbSNP
rs1471477760	1096	dbSNP
rs1407035832	1097	dbSNP
rs1328536822	1099	dbSNP
rs770345743	1122	dbSNP
rs549690244	1128	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.