ID	Duplex structure	Position	Score	MFE
1	miRNA 3' guGGGGGU-CUCGGA-GUCGAGGGGu 5' |:|||| :|| | ||||||| Target 5' cgCTCCCACTCTTCTCCTGCTCCCCc 3'	461 - 486	154.00	-28.50
2	miRNA 3' guGGGGGUCUCGGAGUCGAGGGgu 5' |||||: :|| | |||||| Target 5' tgCCCCCGCGGCAACCGCTCCCac 3'	10 - 33	150.00	-29.10
3	miRNA 3' guGGGGGUC--UCGGAGUCGA---GGGGu 5' | |:: | |||| |:||| |||| Target 5' ccCACTTTGCTAGCC-CGGCTGTGCCCCt 3'	39 - 66	111.00	-22.90

Mutant ID	Mutant Position	Mutant Source
COSN30507675	4	COSMIC
COSN30144027	18	COSMIC
COSN30651368	20	COSMIC
COSN20061724	35	COSMIC
COSN24382250	37	COSMIC
COSN31503117	41	COSMIC
COSN30162354	73	COSMIC
COSN1799214	104	COSMIC
COSN9863436	152	COSMIC
COSN7109958	292	COSMIC
COSN32062684	451	COSMIC
COSN16814252	676	COSMIC
COSN31531168	691	COSMIC
COSN26569569	849	COSMIC
COSN30160435	849	COSMIC
COSN31609676	862	COSMIC
COSN26491090	883	COSMIC
COSN31563360	895	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs781019702	1	dbSNP
rs758471186	3	dbSNP
rs1360587883	4	dbSNP
rs1299571537	5	dbSNP
rs1422228444	8	dbSNP
rs1294123584	11	dbSNP
rs750514300	12	dbSNP
rs1165080788	17	dbSNP
rs1425304818	19	dbSNP
rs1417191176	25	dbSNP
rs765542325	26	dbSNP
rs1478147702	27	dbSNP
rs1265257003	32	dbSNP
rs1220269074	33	dbSNP
rs375925488	40	dbSNP
rs146577311	42	dbSNP
rs1224105684	43	dbSNP
rs1306103244	46	dbSNP
rs913420859	54	dbSNP
rs1462281764	55	dbSNP
rs990316811	58	dbSNP
rs1462711358	59	dbSNP
rs1204479126	61	dbSNP
rs558125441	63	dbSNP
rs1471990131	64	dbSNP
rs1179360587	65	dbSNP
rs1409323262	78	dbSNP
rs1419062717	79	dbSNP
rs1156310281	80	dbSNP
rs1044518454	84	dbSNP
rs1166567980	99	dbSNP
rs187540686	101	dbSNP
rs565912474	105	dbSNP
rs549086756	106	dbSNP
rs528950075	116	dbSNP
rs1222074616	117	dbSNP
rs747758298	122	dbSNP
rs780585740	123	dbSNP
rs1285127346	124	dbSNP
rs895487538	128	dbSNP
rs569751277	130	dbSNP
rs1018896774	131	dbSNP
rs184355526	136	dbSNP
rs1005240395	147	dbSNP
rs532948570	153	dbSNP
rs1440956455	156	dbSNP
rs1207046434	157	dbSNP
rs1233887284	158	dbSNP
rs1187916598	161	dbSNP
rs1180578802	165	dbSNP
rs1468699026	166	dbSNP
rs76598083	175	dbSNP
rs994656133	198	dbSNP
rs1422596637	207	dbSNP
rs1219584525	208	dbSNP
rs902733953	215	dbSNP
rs1042614158	218	dbSNP
rs1407786202	223	dbSNP
rs1304832228	226	dbSNP
rs894909983	233	dbSNP
rs1346110932	236	dbSNP
rs76233875	239	dbSNP
rs1216398535	245	dbSNP
rs1274395317	250	dbSNP
rs1063728	251	dbSNP
rs906232964	262	dbSNP
rs1297436577	263	dbSNP
rs1042044060	264	dbSNP
rs915352049	265	dbSNP
rs11575891	266	dbSNP
rs1307230377	274	dbSNP
rs561978004	282	dbSNP
rs542424761	286	dbSNP
rs1203492698	291	dbSNP
rs975245705	300	dbSNP
rs946316145	302	dbSNP
rs913462374	305	dbSNP
rs990734785	315	dbSNP
rs1244547739	333	dbSNP
rs1476113169	335	dbSNP
rs1324326519	336	dbSNP
rs936147678	337	dbSNP
rs1168083347	338	dbSNP
rs375027614	341	dbSNP
rs1418384790	343	dbSNP
rs1362211994	349	dbSNP
rs1427545220	354	dbSNP
rs542537967	355	dbSNP
rs1366096864	358	dbSNP
rs1436268829	371	dbSNP
rs1348913265	372	dbSNP
rs1170792372	379	dbSNP
rs922025736	380	dbSNP
rs865797033	388	dbSNP
rs1341772046	392	dbSNP
rs974839437	402	dbSNP
rs1167838725	420	dbSNP
rs1454670850	425	dbSNP
rs576412920	428	dbSNP
rs1448179159	432	dbSNP
rs1193327623	433	dbSNP
rs1018931301	434	dbSNP
rs1447344451	437	dbSNP
rs1185193211	438	dbSNP
rs973231877	453	dbSNP
rs1233652985	454	dbSNP
rs1423781831	455	dbSNP
rs1206905132	457	dbSNP
rs1340055418	459	dbSNP
rs983454785	467	dbSNP
rs963529428	468	dbSNP
rs1402493540	469	dbSNP
rs950711278	472	dbSNP
rs1235596991	473	dbSNP
rs1022771179	478	dbSNP
rs1013100588	479	dbSNP
rs1299873667	479	dbSNP
rs1383922096	481	dbSNP
rs1244550581	483	dbSNP
rs563009528	485	dbSNP
rs545869832	486	dbSNP
rs187304017	487	dbSNP
rs558064048	488	dbSNP
rs1185194608	489	dbSNP
rs1365444308	489	dbSNP
rs531538427	489	dbSNP
rs1159216391	490	dbSNP
rs534952692	496	dbSNP
rs1387918249	500	dbSNP
rs1455727599	503	dbSNP
rs1411308454	504	dbSNP
rs1338894568	507	dbSNP
rs572389331	508	dbSNP
rs946935466	527	dbSNP
rs1395783283	530	dbSNP
rs1311097701	535	dbSNP
rs1338042304	541	dbSNP
rs1240303324	545	dbSNP
rs895019612	556	dbSNP
rs1033504858	562	dbSNP
rs1288284211	578	dbSNP
rs781544008	581	dbSNP
rs893984388	588	dbSNP
rs182899938	603	dbSNP
rs757415525	605	dbSNP
rs555575669	607	dbSNP
rs906262337	610	dbSNP
rs1042464167	611	dbSNP
rs911967924	615	dbSNP
rs987470432	617	dbSNP
rs1181295292	618	dbSNP
rs929288608	629	dbSNP
rs1432322572	637	dbSNP
rs945031236	638	dbSNP
rs1179130898	641	dbSNP
rs1052581	644	dbSNP
rs1054672147	645	dbSNP
rs1211649484	647	dbSNP
rs1486735170	648	dbSNP
rs1254758005	649	dbSNP
rs569607548	652	dbSNP
rs1317831844	653	dbSNP
rs973738720	654	dbSNP
rs1309628181	655	dbSNP
rs936257349	656	dbSNP
rs1307916989	660	dbSNP
rs963304868	660	dbSNP
rs1349225606	663	dbSNP
rs139805958	668	dbSNP
rs1406224145	674	dbSNP
rs1283434715	683	dbSNP
rs75130874	684	dbSNP
rs370455954	688	dbSNP
rs1252718280	690	dbSNP
rs1023225588	696	dbSNP
rs14924	697	dbSNP
rs115336731	700	dbSNP
rs1490835808	701	dbSNP
rs944817781	715	dbSNP
rs911959003	718	dbSNP
rs1268062646	725	dbSNP
rs1035437859	726	dbSNP
rs1300940257	727	dbSNP
rs111984662	730	dbSNP
rs902838516	732	dbSNP
rs1462788439	733	dbSNP
rs1021276679	737	dbSNP
rs1161079941	740	dbSNP
rs1407371489	744	dbSNP
rs764195270	750	dbSNP
rs1304288597	752	dbSNP
rs1366221600	777	dbSNP
rs1384595166	791	dbSNP
rs1297230352	796	dbSNP
rs983871629	797	dbSNP
rs1227373873	799	dbSNP
rs1272291208	807	dbSNP
rs796782360	810	dbSNP
rs1223213229	814	dbSNP
rs1264772872	819	dbSNP
rs80339062	820	dbSNP
rs1189509230	826	dbSNP
rs1007722783	829	dbSNP
rs1450260703	830	dbSNP
rs1188998299	834	dbSNP
rs1388775431	836	dbSNP
rs973377871	837	dbSNP
rs1426928968	845	dbSNP
rs959261870	847	dbSNP
rs1349775101	849	dbSNP
rs890562351	849	dbSNP
rs1476196118	850	dbSNP
rs1254178624	855	dbSNP
rs75757408	856	dbSNP
rs1290060575	857	dbSNP
rs1385231992	865	dbSNP
rs1033535937	866	dbSNP
rs1294931884	867	dbSNP
rs1384787304	871	dbSNP
rs1486465368	871	dbSNP
rs1263054259	886	dbSNP
rs1207208233	889	dbSNP
rs929362210	894	dbSNP
rs1328975809	899	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.