ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ugcAGUGACGCAAGAGAGGGGc 5' ||||||:| ||||||| Target 5' ttcTCACTGTG-AATCTCCCCg 3'	389 - 409	162.00	-28.50
2	miRNA 3' ugcagugacgcaagAGAGGGgc 5' |||||| Target 5' agcctaccaccaggTCTCCCac 3'	609 - 630	120.00	-13.40
3	miRNA 3' ugCAGUGACGCAAGAGAGGGGc 5' | | || | ||||:||: Target 5' agGCCCCTCCCGCCTCTTCCTg 3'	262 - 283	120.00	-15.80

Mutant ID	Mutant Position	Mutant Source
COSN26596481	139	COSMIC
COSN30110560	165	COSMIC
COSN30134596	290	COSMIC
COSN28689337	444	COSMIC
COSN30125401	477	COSMIC
COSN17956406	485	COSMIC
COSN15624483	493	COSMIC
COSN15624484	603	COSMIC
COSN31556682	661	COSMIC
COSN31602542	696	COSMIC
COSN31601113	709	COSMIC
COSN24306164	736	COSMIC
COSN30544406	757	COSMIC
COSN31597119	761	COSMIC
COSN30532270	764	COSMIC
COSN30134557	822	COSMIC
COSN31553464	832	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1233306388	3	dbSNP
rs201398249	4	dbSNP
rs968539690	5	dbSNP
rs1372522800	11	dbSNP
rs1474559959	12	dbSNP
rs1331795508	13	dbSNP
rs1378760110	17	dbSNP
rs1234014310	18	dbSNP
rs781489763	19	dbSNP
rs575656410	20	dbSNP
rs769891608	21	dbSNP
rs1302283983	22	dbSNP
rs1363684348	26	dbSNP
rs777778959	27	dbSNP
rs749344450	28	dbSNP
rs771465315	30	dbSNP
rs774831421	31	dbSNP
rs1325037104	32	dbSNP
rs1461519694	34	dbSNP
rs1271627233	35	dbSNP
rs759999898	36	dbSNP
rs1367828287	38	dbSNP
rs866419142	39	dbSNP
rs1156306465	45	dbSNP
rs1037013108	52	dbSNP
rs1190434480	55	dbSNP
rs1437871914	61	dbSNP
rs1248678530	65	dbSNP
rs898116236	66	dbSNP
rs1200211408	67	dbSNP
rs1033981865	71	dbSNP
rs773349563	74	dbSNP
rs1210636298	78	dbSNP
rs991347084	81	dbSNP
rs998178692	83	dbSNP
rs1030470039	95	dbSNP
rs541430004	97	dbSNP
rs1184010168	100	dbSNP
rs374245376	102	dbSNP
rs1370107986	105	dbSNP
rs1304605510	106	dbSNP
rs1441260033	107	dbSNP
rs1363965538	108	dbSNP
rs1318008458	109	dbSNP
rs1369933617	116	dbSNP
rs1454852394	117	dbSNP
rs1345750749	118	dbSNP
rs1024215349	123	dbSNP
rs1162554354	125	dbSNP
rs561855842	126	dbSNP
rs1194026680	129	dbSNP
rs1467237129	130	dbSNP
rs1246400527	131	dbSNP
rs971003714	135	dbSNP
rs982415793	138	dbSNP
rs1329333595	141	dbSNP
rs527376620	142	dbSNP
rs1453601063	143	dbSNP
rs1336580147	144	dbSNP
rs941122036	145	dbSNP
rs1339054244	146	dbSNP
rs1244748297	147	dbSNP
rs1294902314	150	dbSNP
rs1032006117	159	dbSNP
rs1361972333	162	dbSNP
rs377630513	166	dbSNP
rs1445820067	169	dbSNP
rs959946620	171	dbSNP
rs992713512	172	dbSNP
rs1388554849	173	dbSNP
rs1172726049	174	dbSNP
rs1339549242	174	dbSNP
rs1429321943	179	dbSNP
rs915716483	181	dbSNP
rs948573888	183	dbSNP
rs1219945829	184	dbSNP
rs1258767741	185	dbSNP
rs983817365	185	dbSNP
rs973880172	186	dbSNP
rs1255561526	188	dbSNP
rs963329698	190	dbSNP
rs1229232378	192	dbSNP
rs1307715042	193	dbSNP
rs1316948861	196	dbSNP
rs939679911	199	dbSNP
rs1258061365	202	dbSNP
rs921416564	204	dbSNP
rs1440480925	206	dbSNP
rs1036660313	212	dbSNP
rs932877183	214	dbSNP
rs1186824904	220	dbSNP
rs1242646601	221	dbSNP
rs1478724525	225	dbSNP
rs900842203	227	dbSNP
rs1051338148	229	dbSNP
rs1462720779	233	dbSNP
rs1368089126	235	dbSNP
rs541269091	236	dbSNP
rs1162791417	238	dbSNP
rs564280606	239	dbSNP
rs891389457	240	dbSNP
rs889267115	254	dbSNP
rs1471890259	257	dbSNP
rs973042436	258	dbSNP
rs1254504380	261	dbSNP
rs1196717786	264	dbSNP
rs72634003	266	dbSNP
rs1043157324	271	dbSNP
rs904648216	273	dbSNP
rs1042430070	283	dbSNP
rs1031640227	295	dbSNP
rs960327886	295	dbSNP
rs1014103991	296	dbSNP
rs1308938478	296	dbSNP
rs1390325377	305	dbSNP
rs903906908	306	dbSNP
rs1463096311	307	dbSNP
rs1174501763	314	dbSNP
rs1001343387	315	dbSNP
rs1158520181	322	dbSNP
rs1033793159	325	dbSNP
rs969852975	326	dbSNP
rs566127095	327	dbSNP
rs1012711182	329	dbSNP
rs1266246064	330	dbSNP
rs1191422383	332	dbSNP
rs1024137056	334	dbSNP
rs1398645493	336	dbSNP
rs971306859	344	dbSNP
rs1207526396	347	dbSNP
rs1327022774	349	dbSNP
rs909709061	351	dbSNP
rs982386037	356	dbSNP
rs1406410577	358	dbSNP
rs1015664370	360	dbSNP
rs1320032594	363	dbSNP
rs961118968	366	dbSNP
rs962431013	370	dbSNP
rs1223817075	374	dbSNP
rs1257385469	375	dbSNP
rs1342365855	378	dbSNP
rs1199667481	379	dbSNP
rs1334147608	381	dbSNP
rs1187418662	408	dbSNP
rs569929320	409	dbSNP
rs1489243083	410	dbSNP
rs921058761	411	dbSNP
rs932769609	415	dbSNP
rs1236194993	416	dbSNP
rs986966966	420	dbSNP
rs1191387665	421	dbSNP
rs1262616366	422	dbSNP
rs912794207	426	dbSNP
rs1328335707	429	dbSNP
rs1319059849	433	dbSNP
rs529322339	438	dbSNP
rs1042689421	442	dbSNP
rs1376139755	449	dbSNP
rs1175939872	451	dbSNP
rs1465669536	452	dbSNP
rs111245437	454	dbSNP
rs1395309788	458	dbSNP
rs936724546	460	dbSNP
rs1159536528	464	dbSNP
rs1167504433	465	dbSNP
rs1043210849	466	dbSNP
rs904699329	467	dbSNP
rs998923906	470	dbSNP
rs1055610675	471	dbSNP
rs1418454287	474	dbSNP
rs114468879	475	dbSNP
rs1339353656	477	dbSNP
rs1485838422	479	dbSNP
rs1386859514	484	dbSNP
rs1302678052	485	dbSNP
rs1329027529	489	dbSNP
rs1213099765	491	dbSNP
rs1232014211	495	dbSNP
rs1012680438	498	dbSNP
rs1024105959	500	dbSNP
rs1355543450	503	dbSNP
rs1339254179	508	dbSNP
rs907075317	509	dbSNP
rs566211265	511	dbSNP
rs1315272764	517	dbSNP
rs1214445103	520	dbSNP
rs1415793988	521	dbSNP
rs1370398754	531	dbSNP
rs534797730	548	dbSNP
rs1490475840	549	dbSNP
rs1015552785	550	dbSNP
rs1398163425	551	dbSNP
rs3215740	554	dbSNP
rs1265304475	557	dbSNP
rs1415590526	558	dbSNP
rs1189908284	565	dbSNP
rs1432873885	566	dbSNP
rs557790384	567	dbSNP
rs969908888	569	dbSNP
rs1250850149	574	dbSNP
rs1005432033	580	dbSNP
rs1438209782	583	dbSNP
rs1273923867	584	dbSNP
rs1209318604	586	dbSNP
rs1016738396	588	dbSNP
rs1295186983	590	dbSNP
rs1234511139	597	dbSNP
rs1350379454	598	dbSNP
rs1278200875	600	dbSNP
rs764472835	604	dbSNP
rs1444757642	606	dbSNP
rs571511932	609	dbSNP
rs1404259375	611	dbSNP
rs972490610	615	dbSNP
rs571773227	616	dbSNP
rs1459862798	617	dbSNP
rs1166425118	623	dbSNP
rs1461940407	624	dbSNP
rs1411078046	628	dbSNP
rs34644105	630	dbSNP
rs1470999047	632	dbSNP
rs1294794673	639	dbSNP
rs953792311	643	dbSNP
rs1467309937	644	dbSNP
rs986937291	644	dbSNP
rs73976190	648	dbSNP
rs1329107486	651	dbSNP
rs1282723044	652	dbSNP
rs1225837666	653	dbSNP
rs1331604619	655	dbSNP
rs966890795	658	dbSNP
rs1274618113	661	dbSNP
rs985166359	662	dbSNP
rs978747499	663	dbSNP
rs1327358658	664	dbSNP
rs910896095	666	dbSNP
rs1387819409	667	dbSNP
rs925591943	668	dbSNP
rs1408523414	678	dbSNP
rs1322286594	681	dbSNP
rs369296924	683	dbSNP
rs1263801699	687	dbSNP
rs1055151565	688	dbSNP
rs1452865107	689	dbSNP
rs916624909	690	dbSNP
rs1194378354	691	dbSNP
rs557248334	693	dbSNP
rs934849634	701	dbSNP
rs757473804	702	dbSNP
rs1053196992	703	dbSNP
rs907044323	706	dbSNP
rs950139752	709	dbSNP
rs1305084666	713	dbSNP
rs1044340736	715	dbSNP
rs1360730396	715	dbSNP
rs1004038612	718	dbSNP
rs1286603944	727	dbSNP
rs1386201208	747	dbSNP
rs573981425	751	dbSNP
rs1163251164	756	dbSNP
rs1005400998	757	dbSNP
rs535464094	767	dbSNP
rs1449079538	769	dbSNP
rs555067871	773	dbSNP
rs995555127	776	dbSNP
rs539192477	777	dbSNP
rs116213695	778	dbSNP
rs1237372775	779	dbSNP
rs1209182162	780	dbSNP
rs1486823370	781	dbSNP
rs953720546	782	dbSNP
rs955071423	787	dbSNP
rs1234652767	789	dbSNP
rs1306524336	795	dbSNP
rs985218311	796	dbSNP
rs1007961705	797	dbSNP
rs1444065536	798	dbSNP
rs540770401	803	dbSNP
rs1292532298	809	dbSNP
rs966859720	816	dbSNP
rs146467608	821	dbSNP
rs978291436	824	dbSNP
rs1377106160	830	dbSNP
rs925503287	838	dbSNP
rs1017927336	842	dbSNP
rs1461709305	847	dbSNP
rs958328111	851	dbSNP
rs991508144	853	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.