ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ucGAAGGACGAG-GGACGAGAUGu 5' | |||||:|| ||| |||||| Target 5' ggCCTCCTGTTCGCCTCCTCTACa 3'	99 - 122	153.00	-28.70
2	miRNA 3' ucgaagGACGAGGGACGAGAUGu 5' :||: || |||||:| Target 5' aggtgaTTGTGCCAGGCTCTGCg 3'	1155 - 1177	137.00	-21.10
3	miRNA 3' ucGAAG-GACGA----GGGACGAGAugu 5' |||| ||| | : :|||||| Target 5' ggCTTCACTGGTTGAGTGTTGCTCTcta 3'	154 - 181	119.00	-17.40

Mutant ID	Mutant Position	Mutant Source
1192709	92	ClinVar
1192710	93	ClinVar
COSN32053071	36	COSMIC
COSN31559065	48	COSMIC
COSN30166271	53	COSMIC
COSN31502234	82	COSMIC
COSN28188649	92	COSMIC
COSN27339884	94	COSMIC
COSN31525250	111	COSMIC
COSN13414896	112	COSMIC
COSN30102165	113	COSMIC
COSN30159237	114	COSMIC
COSN30462311	117	COSMIC
COSN31494160	118	COSMIC
COSN30152228	119	COSMIC
COSN5718622	151	COSMIC
COSN30124375	205	COSMIC
COSN8541029	215	COSMIC
COSN31599908	255	COSMIC
COSN1151296	258	COSMIC
COSN7378819	454	COSMIC
COSN24645798	471	COSMIC
COSN5951075	501	COSMIC
COSN1582529	529	COSMIC
COSN30076072	742	COSMIC
COSN22558627	776	COSMIC
COSN31960744	820	COSMIC
COSN25722546	1441	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs200946562	4	dbSNP
rs202082987	6	dbSNP
rs373819744	7	dbSNP
rs992264830	9	dbSNP
rs782315420	10	dbSNP
rs200466635	13	dbSNP
rs782159669	14	dbSNP
rs1486871391	19	dbSNP
rs782690098	26	dbSNP
rs781930565	28	dbSNP
rs782741280	37	dbSNP
rs377328068	38	dbSNP
rs781854190	41	dbSNP
rs782517618	42	dbSNP
rs1346272073	46	dbSNP
rs372481939	50	dbSNP
rs782296707	51	dbSNP
rs200909686	52	dbSNP
rs782576628	53	dbSNP
rs202197363	54	dbSNP
rs1416808626	65	dbSNP
rs767541851	66	dbSNP
rs1413923895	67	dbSNP
rs200464962	71	dbSNP
rs1169548473	74	dbSNP
rs16932921	75	dbSNP
rs1371952238	80	dbSNP
rs199732427	81	dbSNP
rs200182933	82	dbSNP
rs56151134	84	dbSNP
rs113726594	88	dbSNP
rs1187752889	88	dbSNP
rs201020993	88	dbSNP
rs1253341141	90	dbSNP
rs56321187	91	dbSNP
rs879986500	91	dbSNP
rs7136817	92	dbSNP
rs7307451	93	dbSNP
rs1249619838	100	dbSNP
rs1235381314	101	dbSNP
rs1335797682	107	dbSNP
rs1292897317	111	dbSNP
rs199774792	112	dbSNP
rs1338466204	113	dbSNP
rs782140051	117	dbSNP
rs933433183	119	dbSNP
rs1330844642	122	dbSNP
rs782525558	126	dbSNP
rs139368230	127	dbSNP
rs200708593	128	dbSNP
rs201639799	130	dbSNP
rs199531126	134	dbSNP
rs1395149955	142	dbSNP
rs889125054	143	dbSNP
rs200665617	145	dbSNP
rs1462548276	150	dbSNP
rs1418171146	151	dbSNP
rs1472027188	152	dbSNP
rs201985731	154	dbSNP
rs200091027	155	dbSNP
rs904688281	158	dbSNP
rs200929851	162	dbSNP
rs201738192	164	dbSNP
rs200061207	176	dbSNP
rs1272842826	182	dbSNP
rs201200230	187	dbSNP
rs1354036836	189	dbSNP
rs370677099	190	dbSNP
rs202147012	193	dbSNP
rs1030739333	198	dbSNP
rs782299603	200	dbSNP
rs879947870	203	dbSNP
rs118185785	205	dbSNP
rs1239698257	206	dbSNP
rs199857555	214	dbSNP
rs1306987701	229	dbSNP
rs201013016	237	dbSNP
rs1442525465	243	dbSNP
rs202225388	248	dbSNP
rs1394510839	251	dbSNP
rs3213427	253	dbSNP
rs1387608833	255	dbSNP
rs201058836	260	dbSNP
rs782246469	267	dbSNP
rs1159563443	268	dbSNP
rs1469941923	274	dbSNP
rs1379042568	280	dbSNP
rs28919580	286	dbSNP
rs781957203	298	dbSNP
rs1448898666	300	dbSNP
rs1285204186	302	dbSNP
rs200271394	303	dbSNP
rs536098751	306	dbSNP
rs201618825	315	dbSNP
rs199727997	328	dbSNP
rs1283824337	330	dbSNP
rs200636893	333	dbSNP
rs1236354894	341	dbSNP
rs35548287	342	dbSNP
rs1301985961	344	dbSNP
rs1433653641	346	dbSNP
rs199739909	347	dbSNP
rs200816523	355	dbSNP
rs1327898046	357	dbSNP
rs868961142	358	dbSNP
rs201627409	382	dbSNP
rs199875333	386	dbSNP
rs1400544739	387	dbSNP
rs200664387	388	dbSNP
rs919114009	390	dbSNP
rs1435816217	401	dbSNP
rs186463069	409	dbSNP
rs3213428	411	dbSNP
rs1476430587	412	dbSNP
rs1242956618	416	dbSNP
rs1192679583	418	dbSNP
rs3213429	419	dbSNP
rs202028146	431	dbSNP
rs200058720	445	dbSNP
rs143745546	458	dbSNP
rs1212951979	465	dbSNP
rs1348570620	472	dbSNP
rs201877444	478	dbSNP
rs1277820794	486	dbSNP
rs782352347	489	dbSNP
rs200196003	492	dbSNP
rs139621602	499	dbSNP
rs201379343	499	dbSNP
rs1338911480	500	dbSNP
rs28990968	500	dbSNP
rs1315113490	501	dbSNP
rs34744701	513	dbSNP
rs1375443815	518	dbSNP
rs3829972	524	dbSNP
rs1411750797	525	dbSNP
rs1420266014	551	dbSNP
rs200561797	558	dbSNP
rs1167560953	561	dbSNP
rs35753068	572	dbSNP
rs201448594	573	dbSNP
rs1476930796	580	dbSNP
rs55749318	585	dbSNP
rs999131922	593	dbSNP
rs1485856714	594	dbSNP
rs1236882556	605	dbSNP
rs1207877875	608	dbSNP
rs539803066	608	dbSNP
rs56105558	608	dbSNP
rs199630739	613	dbSNP
rs782099376	618	dbSNP
rs868964155	620	dbSNP
rs200605097	633	dbSNP
rs1217656497	640	dbSNP
rs201413896	647	dbSNP
rs1315741008	650	dbSNP
rs191336635	665	dbSNP
rs1294742926	668	dbSNP
rs1234329892	669	dbSNP
rs1378875400	680	dbSNP
rs895990690	713	dbSNP
rs1444907422	724	dbSNP
rs1355198756	725	dbSNP
rs199762428	726	dbSNP
rs1330831253	727	dbSNP
rs181673678	731	dbSNP
rs74059132	732	dbSNP
rs969024139	737	dbSNP
rs1001850396	747	dbSNP
rs200091466	752	dbSNP
rs1016122276	753	dbSNP
rs1481544091	755	dbSNP
rs200883969	756	dbSNP
rs963240826	762	dbSNP
rs1233376771	763	dbSNP
rs1197532639	764	dbSNP
rs1456046826	766	dbSNP
rs1274718473	767	dbSNP
rs201597912	768	dbSNP
rs1213883895	770	dbSNP
rs1321500734	771	dbSNP
rs1310955562	772	dbSNP
rs972007482	773	dbSNP
rs919050584	774	dbSNP
rs1374776109	787	dbSNP
rs1309129059	796	dbSNP
rs954599657	798	dbSNP
rs1444713418	804	dbSNP
rs1396722484	806	dbSNP
rs199911194	808	dbSNP
rs910662165	820	dbSNP
rs200838960	821	dbSNP
rs1349107986	825	dbSNP
rs201496151	828	dbSNP
rs146044918	833	dbSNP
rs1420649637	845	dbSNP
rs200185608	849	dbSNP
rs1408681436	853	dbSNP
rs1177910660	855	dbSNP
rs782055101	858	dbSNP
rs1437304142	868	dbSNP
rs1254260281	877	dbSNP
rs1193892364	885	dbSNP
rs201120419	890	dbSNP
rs1487986702	896	dbSNP
rs1263308617	901	dbSNP
rs28990969	909	dbSNP
rs200488375	916	dbSNP
rs201111757	917	dbSNP
rs202143153	923	dbSNP
rs1304184696	924	dbSNP
rs1365844370	931	dbSNP
rs1455199971	933	dbSNP
rs28990970	937	dbSNP
rs200159175	939	dbSNP
rs1360878787	946	dbSNP
rs139558800	949	dbSNP
rs1417719601	950	dbSNP
rs1396796225	956	dbSNP
rs55934918	958	dbSNP
rs200555088	963	dbSNP
rs201438622	964	dbSNP
rs199533028	967	dbSNP
rs200793677	971	dbSNP
rs201513292	972	dbSNP
rs199802443	975	dbSNP
rs1486414614	976	dbSNP
rs1262839712	978	dbSNP
rs116826563	985	dbSNP
rs781782957	986	dbSNP
rs1232682369	1014	dbSNP
rs1310498870	1016	dbSNP
rs1298032767	1018	dbSNP
rs7901	1019	dbSNP
rs200085451	1020	dbSNP
rs201242948	1022	dbSNP
rs202053313	1027	dbSNP
rs199821877	1058	dbSNP
rs1297411864	1061	dbSNP
rs1397903781	1070	dbSNP
rs200951019	1071	dbSNP
rs1173069007	1074	dbSNP
rs185853332	1078	dbSNP
rs1465127014	1079	dbSNP
rs1375387215	1091	dbSNP
rs202131652	1097	dbSNP
rs200460560	1099	dbSNP
rs1422456000	1100	dbSNP
rs1192036492	1106	dbSNP
rs1486840342	1107	dbSNP
rs200903724	1112	dbSNP
rs1200341078	1113	dbSNP
rs1026268102	1115	dbSNP
rs202055827	1131	dbSNP
rs987350038	1132	dbSNP
rs782663370	1133	dbSNP
rs200407231	1136	dbSNP
rs1317588841	1138	dbSNP
rs1276502757	1139	dbSNP
rs1045261	1143	dbSNP
rs1357075726	1148	dbSNP
rs1395315742	1154	dbSNP
rs1399454820	1155	dbSNP
rs1299443470	1156	dbSNP
rs1465687122	1157	dbSNP
rs1422525956	1159	dbSNP
rs1168073440	1163	dbSNP
rs1476406306	1164	dbSNP
rs199777877	1165	dbSNP
rs149728907	1166	dbSNP
rs201150644	1177	dbSNP
rs199770820	1178	dbSNP
rs1457351523	1182	dbSNP
rs1256423332	1183	dbSNP
rs1215744418	1188	dbSNP
rs1336677264	1195	dbSNP
rs1284510958	1196	dbSNP
rs1243782026	1199	dbSNP
rs1338700515	1206	dbSNP
rs1309022024	1211	dbSNP
rs989466640	1224	dbSNP
rs1446947693	1233	dbSNP
rs1398866484	1239	dbSNP
rs1328916799	1244	dbSNP
rs1462635340	1247	dbSNP
rs200698109	1254	dbSNP
rs201347732	1255	dbSNP
rs917531607	1256	dbSNP
rs1163106041	1263	dbSNP
rs199527207	1286	dbSNP
rs950253265	1287	dbSNP
rs1044514900	1289	dbSNP
rs1456682332	1296	dbSNP
rs781950482	1300	dbSNP
rs1366102030	1301	dbSNP
rs1179758459	1309	dbSNP
rs143986210	1315	dbSNP
rs906043302	1317	dbSNP
rs1236360912	1320	dbSNP
rs782326475	1322	dbSNP
rs201757201	1336	dbSNP
rs200132339	1337	dbSNP
rs1195746133	1342	dbSNP
rs1452468968	1352	dbSNP
rs1209063773	1353	dbSNP
rs200551333	1359	dbSNP
rs937727777	1362	dbSNP
rs1288109363	1365	dbSNP
rs1037447689	1366	dbSNP
rs1375528872	1371	dbSNP
rs1442455071	1372	dbSNP
rs1331365306	1375	dbSNP
rs1322442782	1384	dbSNP
rs1385471154	1385	dbSNP
rs1387545944	1389	dbSNP
rs1159788581	1392	dbSNP
rs190312260	1399	dbSNP
rs993199670	1405	dbSNP
rs1175679978	1406	dbSNP
rs1478379783	1416	dbSNP
rs28990971	1418	dbSNP
rs1196966000	1422	dbSNP
rs890348119	1425	dbSNP
rs200021012	1433	dbSNP
rs201102164	1436	dbSNP
rs1264983741	1439	dbSNP
rs1217268871	1442	dbSNP
rs1352975623	1447	dbSNP
rs28990972	1460	dbSNP
rs202233728	1462	dbSNP
rs1345045134	1467	dbSNP
rs782758376	1469	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.