ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gguuGGAG--CUGCUAGAGGAGu 5' |||| |:| ||||||| Target 5' gagcCCTCTGGGC-CTCTCCTCc 3'	634 - 655	150.00	-17.00
2	miRNA 3' gguuGGAGCUGCUAGAGGAGu 5' || |||| |:||||| Target 5' gttcCCCCGAC--TTTCCTCt 3'	583 - 601	141.00	-15.70
3	miRNA 3' ggUUGGAG-CUGCUAGAGGAgu 5' || | | ||: ||||:| Target 5' caAAGCACAGATCCTCTCTTac 3'	513 - 534	115.00	-8.80

Mutant ID	Mutant Position	Mutant Source
COSN26983885	23	COSMIC
COSN31491180	25	COSMIC
COSN30509438	52	COSMIC
COSN30697361	84	COSMIC
COSN30526957	185	COSMIC
COSN25919225	195	COSMIC
COSN19658384	198	COSMIC
COSN28819643	235	COSMIC
COSN25099701	249	COSMIC
COSN31551559	272	COSMIC
COSN31544870	330	COSMIC
COSN31534926	352	COSMIC
COSN31607882	353	COSMIC
COSN32070987	379	COSMIC
COSN31552165	381	COSMIC
COSN31565173	449	COSMIC
COSN31569964	613	COSMIC
COSN31596354	665	COSMIC
COSN31576888	676	COSMIC
COSN31543806	696	COSMIC
COSN31488571	698	COSMIC
COSN31550388	717	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs769725251	1	dbSNP
rs773494895	8	dbSNP
rs1180244056	9	dbSNP
rs376318609	11	dbSNP
rs766700751	12	dbSNP
rs984202636	16	dbSNP
rs1236587528	24	dbSNP
rs752008529	27	dbSNP
rs759957608	28	dbSNP
rs1223579728	29	dbSNP
rs1294221956	31	dbSNP
rs768159817	33	dbSNP
rs368499478	35	dbSNP
rs1251841028	36	dbSNP
rs756793690	37	dbSNP
rs764596364	38	dbSNP
rs1180099137	40	dbSNP
rs749936225	42	dbSNP
rs758113629	49	dbSNP
rs779654574	52	dbSNP
rs1242755050	66	dbSNP
rs529923928	67	dbSNP
rs1316446406	72	dbSNP
rs564601061	79	dbSNP
rs1222335426	102	dbSNP
rs1390108263	111	dbSNP
rs1303459305	115	dbSNP
rs866430606	115	dbSNP
rs935361827	116	dbSNP
rs1373845838	122	dbSNP
rs1160296170	125	dbSNP
rs1420087427	134	dbSNP
rs1379393996	139	dbSNP
rs1156802072	141	dbSNP
rs988608844	145	dbSNP
rs901057130	148	dbSNP
rs1180751495	150	dbSNP
rs915354968	161	dbSNP
rs1216795146	168	dbSNP
rs547997540	170	dbSNP
rs1197101910	173	dbSNP
rs1267905502	178	dbSNP
rs1472765320	182	dbSNP
rs1237560749	183	dbSNP
rs1398679875	183	dbSNP
rs563115855	184	dbSNP
rs761406180	184	dbSNP
rs1227795769	185	dbSNP
rs1300296631	185	dbSNP
rs1359700520	185	dbSNP
rs368133035	185	dbSNP
rs1286872370	186	dbSNP
rs1459427240	186	dbSNP
rs1368976790	187	dbSNP
rs1473939766	188	dbSNP
rs1193960704	189	dbSNP
rs144068607	191	dbSNP
rs1487732899	191	dbSNP
rs1332794751	192	dbSNP
rs369788126	192	dbSNP
rs1049675132	193	dbSNP
rs879837727	193	dbSNP
rs1388296937	194	dbSNP
rs796169932	194	dbSNP
rs866747080	194	dbSNP
rs1292877719	195	dbSNP
rs868710070	195	dbSNP
rs1160936921	196	dbSNP
rs1405291585	197	dbSNP
rs1459762262	198	dbSNP
rs866184672	198	dbSNP
rs1399635559	199	dbSNP
rs1164833994	200	dbSNP
rs1191568633	200	dbSNP
rs1352156799	201	dbSNP
rs999848574	201	dbSNP
rs550211469	203	dbSNP
rs1031391843	204	dbSNP
rs1199337728	205	dbSNP
rs1260133806	205	dbSNP
rs1034477815	206	dbSNP
rs1235613162	206	dbSNP
rs1331490271	206	dbSNP
rs1300284329	211	dbSNP
rs1351501650	211	dbSNP
rs1218403250	213	dbSNP
rs1378993023	213	dbSNP
rs1438670938	213	dbSNP
rs1005468141	214	dbSNP
rs1464644352	215	dbSNP
rs371154329	219	dbSNP
rs1466196362	224	dbSNP
rs571642110	228	dbSNP
rs1009697400	235	dbSNP
rs1179742210	237	dbSNP
rs1240571330	237	dbSNP
rs1372847122	237	dbSNP
rs1229005266	238	dbSNP
rs1050259664	239	dbSNP
rs1292110410	240	dbSNP
rs888949616	250	dbSNP
rs1323493877	253	dbSNP
rs944590371	263	dbSNP
rs1231785551	264	dbSNP
rs1220182692	265	dbSNP
rs1309890639	272	dbSNP
rs770461787	273	dbSNP
rs1290867995	275	dbSNP
rs1483280319	277	dbSNP
rs150041319	278	dbSNP
rs1230865843	284	dbSNP
rs970916252	286	dbSNP
rs1401039100	289	dbSNP
rs1173481561	294	dbSNP
rs897460916	295	dbSNP
rs1382499028	301	dbSNP
rs1469278501	303	dbSNP
rs1182003064	306	dbSNP
rs1470034928	313	dbSNP
rs759927114	314	dbSNP
rs199943083	316	dbSNP
rs981017282	321	dbSNP
rs1033954818	334	dbSNP
rs1415312930	337	dbSNP
rs1027803280	338	dbSNP
rs887569497	340	dbSNP
rs1163510241	341	dbSNP
rs1286418009	344	dbSNP
rs1223187886	352	dbSNP
rs952677101	353	dbSNP
rs984540667	357	dbSNP
rs1005722137	360	dbSNP
rs1339306978	367	dbSNP
rs1023769749	372	dbSNP
rs1310713043	379	dbSNP
rs1401413270	382	dbSNP
rs1299148403	385	dbSNP
rs969554933	393	dbSNP
rs1400685620	398	dbSNP
rs982333531	400	dbSNP
rs547457236	420	dbSNP
rs1306528966	423	dbSNP
rs566389519	427	dbSNP
rs1354236993	433	dbSNP
rs908586231	434	dbSNP
rs112643370	435	dbSNP
rs1286118561	449	dbSNP
rs578112772	456	dbSNP
rs1214793004	462	dbSNP
rs1445281601	476	dbSNP
rs1291946517	477	dbSNP
rs915306554	478	dbSNP
rs1210304342	481	dbSNP
rs536916167	486	dbSNP
rs75775181	488	dbSNP
rs1290855502	496	dbSNP
rs187864696	497	dbSNP
rs1342754148	500	dbSNP
rs1207080013	501	dbSNP
rs1279295540	504	dbSNP
rs1270613702	510	dbSNP
rs1343731002	526	dbSNP
rs1326124627	529	dbSNP
rs1436923027	533	dbSNP
rs1162852976	534	dbSNP
rs1393888079	534	dbSNP
rs932512345	536	dbSNP
rs1424604847	537	dbSNP
rs1049622283	538	dbSNP
rs910356657	539	dbSNP
rs1248194432	555	dbSNP
rs904464605	557	dbSNP
rs1467949171	559	dbSNP
rs1270775579	560	dbSNP
rs944559516	563	dbSNP
rs1318236371	572	dbSNP
rs1040676501	574	dbSNP
rs1360339633	576	dbSNP
rs1053199622	583	dbSNP
rs1442272708	591	dbSNP
rs1467255509	597	dbSNP
rs919210186	601	dbSNP
rs928957031	606	dbSNP
rs1048758775	611	dbSNP
rs1182420832	614	dbSNP
rs1427050130	621	dbSNP
rs1391370068	625	dbSNP
rs1451877575	628	dbSNP
rs1172920322	633	dbSNP
rs1453780562	634	dbSNP
rs576668262	642	dbSNP
rs1180361511	643	dbSNP
rs1457834325	646	dbSNP
rs1231770003	655	dbSNP
rs1202043538	656	dbSNP
rs1024095182	657	dbSNP
rs906680444	657	dbSNP
rs868610893	658	dbSNP
rs1235473408	668	dbSNP
rs1347524194	673	dbSNP
rs775505595	676	dbSNP
rs1301895262	679	dbSNP
rs1419271516	680	dbSNP
rs1374182955	681	dbSNP
rs887445514	684	dbSNP
rs1176611618	685	dbSNP
rs1012639084	689	dbSNP
rs1044183591	694	dbSNP
rs531927581	695	dbSNP
rs1404122788	698	dbSNP
rs905339417	700	dbSNP
rs1003739906	704	dbSNP
rs1484067919	711	dbSNP
rs1035669990	717	dbSNP
rs763299383	718	dbSNP
rs759239190	725	dbSNP
rs764070768	733	dbSNP
rs1268286009	735	dbSNP
rs1242994027	736	dbSNP
rs540780033	737	dbSNP
rs1219546693	742	dbSNP
rs537239587	744	dbSNP
rs1381348507	745	dbSNP
rs968032376	746	dbSNP
rs986154965	747	dbSNP
rs191077534	751	dbSNP
rs761712304	752	dbSNP
rs975975010	754	dbSNP
rs767406468	759	dbSNP
rs1465817128	761	dbSNP
rs1191674366	768	dbSNP
rs922721046	771	dbSNP
rs953853244	778	dbSNP
rs985280172	784	dbSNP
rs929200440	785	dbSNP
rs925832000	788	dbSNP
rs935922763	789	dbSNP
rs1053073734	795	dbSNP
rs1219235920	803	dbSNP
rs1486519966	805	dbSNP
rs1260660789	806	dbSNP
rs1049458191	811	dbSNP
rs1214207001	812	dbSNP
rs1323605862	813	dbSNP
rs14932	815	dbSNP
rs759763276	816	dbSNP
rs949771106	818	dbSNP
rs1427506236	827	dbSNP
rs1353772840	829	dbSNP
rs1279931796	831	dbSNP
rs1382575422	832	dbSNP
rs750382783	836	dbSNP
rs769568858	843	dbSNP
rs1045493964	852	dbSNP
rs1459340061	868	dbSNP
rs182106210	869	dbSNP
rs1163550312	871	dbSNP
rs1044152916	875	dbSNP
rs1002348631	877	dbSNP
rs576842116	878	dbSNP
rs906983113	879	dbSNP
rs1004115782	880	dbSNP
rs541056133	882	dbSNP
rs1193374370	883	dbSNP
rs1213477392	884	dbSNP
rs1429498360	900	dbSNP
rs1261413761	901	dbSNP
rs1185319388	904	dbSNP
rs1055310072	909	dbSNP
rs1273945635	909	dbSNP
rs1332682482	910	dbSNP
rs558889578	910	dbSNP
rs1056594809	911	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.