>HDGF|NM_001126050|3'UTR
   1 CCACCAATGTTTCAAGAGGAGCCCCCACCCTGTTCCTGCTGCTGTCTGGGTGCTACTGGGGAAACTGGCCATGGCCTGCA
  81 AACTGGGAACCCCTTTCCCACCCCAACCTGCTCTCCTCTTCTACTCACTTTTCCCACTCCAAGCCCAGCCCATGGAGATT
 161 GACCTGGATGGGGCAGGCCACCTGGCTCTCACCTCTAGGTCCCCATACTCCTATGATCTGAGTCAGAGCCATGTCTTCTC
 241 CCTGGAATGAGTTGAGGCCACTGTGTTCCTTCCGCTTGGAGCTATTTTCCAGGCTTCTGCTGGGGCCTGGGACAACTGCT
 321 CCCACCTCCTGACACCCTTCTCCCACTCTCCTAGGCATTCTGGACCTCTGGGTTGGGATCAGGGGTAGGAATGGAAAGGA
 401 TGGAGCATCAACAGCAGGGTGGGCTTGTGGGGCCTGGGAGGGGCAATCCTCAAATGCGGGGTGGGGGCAGCACAGGAGGG
 481 CGGCCTCCTTCTGAGCTCCTGTCCCCTGCTACACCTATTATCCCAGCTGCCTAGATTCAGGGAAAGTGGGACAGCTTGTA
 561 GGGGAGGGGCTCCTTTCCATAAATCCTTGATGATTGACAACACCCATTTTTCCTTTTGCCGACCCCAAGAGTTTTGGGAG
 641 TTGTAGTTAATCATCAAGAGAATTTGGGGCTTCCAAGTTGTTCGGGCCAAGGACCTGAGACCTGAAGGGTTGACTTTACC
 721 CATTTGGGTGGGAGTGTTGAGCATCTGTCCCCCTTTAGATCTCTGAAGCCACAAATAGGATGCTTGGGAAGACTCCTAGC
 801 TGTCCTTTTTCCTCTCCACACAGTGCTCAAGGCCAGCTTATAGTCATATATATCACCCAGACATAAAGGAAAAGACACAT
 881 TTTTTAGGAAATGTTTTTAATAAAAGAAAATTACAAAAAAAAATTTTAAAGACCCCTAACCCTTTGTGTGCTCTCCATTC
 961 TGCTCCTTCCCCATCGTTGCCCCCATTTCTGAGGTGCACTGGGAGGCTCCCCTTCTATTTGGGGCTTGATGACTTTCTTT
1041 TTGTAGCTGGGGCTTTGATGTTCCTTCCAGTGTCATTTCTCATCCACATACCCTGACCTGGCCCCCTCAGTGTTGTCACC
1121 AGATCTGATTTGTAACCCACTGAGAGGACAGAGAGAAATAAGTGCCCTCTCCCACCCTCTTCCTACTGGTCTCTCTATGC
1201 CTCTCTACAGTCTCGTCTCTTTTACCCTGGCCCCTCTCCCTTGGGCTCTGATGAAAAATTGCTGACTGTAGCTTTGGAAG
1281 TTTAGCTCTGAGAACCGTAGATGATTTCAGTTCTAGGAAAATAAAACCCGTTGATTACTATAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uuuagGGUA--GG------GGUCCUUGGGg 5' |||| || |::||||||| Target 5' actggCCATGGCCTGCAAACTGGGAACCCc 3'	64 - 93	149.00	-25.30
2	miRNA 3' uuuaGGGUA-GGGG---UCCUUGGGg 5' || || :::| ||||:||| Target 5' -ccaCCAATGTTTCAAGAGGAGCCCc 3'	1 - 25	134.00	-20.70
3	miRNA 3' uuUAGGGUAGGGGUCCUUGGGg 5' |||: ||:: | ||||| Target 5' agATCTGATTT---GTAACCCa 3'	1121 - 1139	121.00	-11.40

Mutant ID	Mutant Position	Mutant Source
COSN30515293	7	COSMIC
COSN20048132	13	COSMIC
COSN30522638	17	COSMIC
COSN31497739	19	COSMIC
COSN26957679	27	COSMIC
COSN28846451	46	COSMIC
COSN31560070	52	COSMIC
COSN30504382	53	COSMIC
COSN20048131	56	COSMIC
COSN30515830	74	COSMIC
COSN31498013	86	COSMIC
COSN20048130	97	COSMIC
COSN20048129	102	COSMIC
COSN30698706	129	COSMIC
COSN30448034	133	COSMIC
COSN20048128	145	COSMIC
COSN20048127	151	COSMIC
COSN32064393	190	COSMIC
COSN1090220	221	COSMIC
COSN30513454	223	COSMIC
COSN20048126	240	COSMIC
COSN19751296	274	COSMIC
COSN31604573	275	COSMIC
COSN31590958	279	COSMIC
COSN20048125	297	COSMIC
COSN20048124	319	COSMIC
COSN20048122	340	COSMIC
COSN20048121	366	COSMIC
COSN21181678	368	COSMIC
COSN20048120	471	COSMIC
COSN16131710	481	COSMIC
COSN21077464	490	COSMIC
COSN20048119	491	COSMIC
COSN30655904	494	COSMIC
COSN22708405	592	COSMIC
COSN31608710	616	COSMIC
COSN20052292	629	COSMIC
COSN31564827	683	COSMIC
COSN16128463	699	COSMIC
COSN32063501	727	COSMIC
COSN26660939	825	COSMIC
COSN4744091	849	COSMIC
COSN30161775	873	COSMIC
COSN31661798	880	COSMIC
COSN20095450	914	COSMIC
COSN31523184	985	COSMIC
COSN31548360	989	COSMIC
COSN31589224	997	COSMIC
COSN1407568	1048	COSMIC
COSN22445060	1127	COSMIC
COSN21373532	1227	COSMIC
COSN26554725	1295	COSMIC
COSN32082890	1317	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs765803425	2	dbSNP
rs1489004484	5	dbSNP
rs11556973	7	dbSNP
rs896281935	8	dbSNP
rs753925386	23	dbSNP
rs764091839	28	dbSNP
rs1014697471	29	dbSNP
rs1477272522	32	dbSNP
rs1259311323	36	dbSNP
rs1416492412	37	dbSNP
rs760950183	40	dbSNP
rs1488527601	41	dbSNP
rs1266474668	44	dbSNP
rs1005001875	51	dbSNP
rs566825660	75	dbSNP
rs1369246614	107	dbSNP
rs887454019	117	dbSNP
rs1452704416	120	dbSNP
rs1288547436	121	dbSNP
rs1338648843	123	dbSNP
rs1278963223	126	dbSNP
rs1402057938	126	dbSNP
rs1048709381	140	dbSNP
rs1227165592	147	dbSNP
rs761518823	152	dbSNP
rs144243169	159	dbSNP
rs1215468134	174	dbSNP
rs1354550131	179	dbSNP
rs1283466787	180	dbSNP
rs11556977	184	dbSNP
rs1468654460	187	dbSNP
rs1406039162	188	dbSNP
rs1040328912	191	dbSNP
rs1183536376	193	dbSNP
rs1414395122	195	dbSNP
rs1481653579	202	dbSNP
rs1418309598	205	dbSNP
rs1362189770	213	dbSNP
rs1406048282	220	dbSNP
rs1421826051	223	dbSNP
rs539607564	224	dbSNP
rs1176905460	229	dbSNP
rs568589896	234	dbSNP
rs550698416	238	dbSNP
rs532200506	240	dbSNP
rs935406720	243	dbSNP
rs1223694313	244	dbSNP
rs768366521	251	dbSNP
rs1262475228	267	dbSNP
rs1240493756	269	dbSNP
rs979409695	271	dbSNP
rs781196137	273	dbSNP
rs749563787	274	dbSNP
rs1359019118	276	dbSNP
rs992464618	277	dbSNP
rs1176696925	292	dbSNP
rs565052677	293	dbSNP
rs1382003508	297	dbSNP
rs1440015704	311	dbSNP
rs1159480053	321	dbSNP
rs540058701	329	dbSNP
rs116207813	332	dbSNP
rs1412856058	348	dbSNP
rs1336298303	351	dbSNP
rs1359780705	356	dbSNP
rs1015139286	358	dbSNP
rs1373635234	360	dbSNP
rs1004673046	366	dbSNP
rs369723450	368	dbSNP
rs866153960	370	dbSNP
rs577504203	371	dbSNP
rs1241240938	386	dbSNP
rs1273904177	387	dbSNP
rs1027303633	399	dbSNP
rs561181123	401	dbSNP
rs1382671187	408	dbSNP
rs1278798632	417	dbSNP
rs1306402252	423	dbSNP
rs995809224	425	dbSNP
rs1338028707	436	dbSNP
rs900153718	456	dbSNP
rs1411982740	457	dbSNP
rs1039957984	458	dbSNP
rs1477185295	460	dbSNP
rs1174528316	461	dbSNP
rs73002713	468	dbSNP
rs1465113483	481	dbSNP
rs574912091	481	dbSNP
rs1401449631	482	dbSNP
rs1329710018	490	dbSNP
rs556376794	490	dbSNP
rs534268126	495	dbSNP
rs1275016711	498	dbSNP
rs1339282012	501	dbSNP
rs1259939914	503	dbSNP
rs1181796469	505	dbSNP
rs1212956130	506	dbSNP
rs1287549090	508	dbSNP
rs1444136774	510	dbSNP
rs1188367772	512	dbSNP
rs544732053	515	dbSNP
rs577276057	517	dbSNP
rs550444571	518	dbSNP
rs1242438081	529	dbSNP
rs1247438982	537	dbSNP
rs1184056621	540	dbSNP
rs199960326	552	dbSNP
rs1425833744	565	dbSNP
rs781239733	576	dbSNP
rs1466667792	582	dbSNP
rs1163178943	597	dbSNP
rs1258686000	607	dbSNP
rs925345802	616	dbSNP
rs757395551	620	dbSNP
rs1339389645	621	dbSNP
rs1397722094	634	dbSNP
rs1279586293	641	dbSNP
rs1340591445	646	dbSNP
rs1244044001	650	dbSNP
rs1282293146	652	dbSNP
rs1243392157	655	dbSNP
rs1287783718	656	dbSNP
rs35554110	661	dbSNP
rs751664551	666	dbSNP
rs1285020601	674	dbSNP
rs1488110706	675	dbSNP
rs777801928	676	dbSNP
rs377077660	683	dbSNP
rs1367196537	684	dbSNP
rs916531893	686	dbSNP
rs1315140795	688	dbSNP
rs1360252195	691	dbSNP
rs1398242978	710	dbSNP
rs1360513948	711	dbSNP
rs1299636854	714	dbSNP
rs992099180	717	dbSNP
rs960902850	729	dbSNP
rs907722145	732	dbSNP
rs1342139111	743	dbSNP
rs11556978	744	dbSNP
rs1308350481	747	dbSNP
rs1431980467	751	dbSNP
rs1351220444	762	dbSNP
rs1238269932	770	dbSNP
rs1288345556	789	dbSNP
rs1486253605	796	dbSNP
rs1199896242	802	dbSNP
rs1254141313	804	dbSNP
rs1444673071	819	dbSNP
rs1380137482	826	dbSNP
rs1438153311	827	dbSNP
rs1362667180	832	dbSNP
rs1380484651	835	dbSNP
rs138665143	836	dbSNP
rs1465125772	841	dbSNP
rs1300612693	845	dbSNP
rs758670127	847	dbSNP
rs1403719359	848	dbSNP
rs1396598659	856	dbSNP
rs1330530682	886	dbSNP
rs951851794	891	dbSNP
rs1161963660	892	dbSNP
rs548570568	900	dbSNP
rs1311770440	903	dbSNP
rs1424091745	906	dbSNP
rs1027376790	910	dbSNP
rs1385881587	911	dbSNP
rs1165575683	912	dbSNP
rs1238029198	914	dbSNP
rs1425444752	916	dbSNP
rs1259623497	922	dbSNP
rs1185695726	923	dbSNP
rs1207135523	924	dbSNP
rs1251657014	924	dbSNP
rs201760421	924	dbSNP
rs60294423	924	dbSNP
rs1173506409	926	dbSNP
rs1378879017	928	dbSNP
rs1486107478	930	dbSNP
rs995841930	934	dbSNP
rs1467101650	940	dbSNP
rs1331839874	941	dbSNP
rs566809290	942	dbSNP
rs1386479705	948	dbSNP
rs1358299414	962	dbSNP
rs1291805034	971	dbSNP
rs1224153128	973	dbSNP
rs554872714	1001	dbSNP
rs1019018275	1009	dbSNP
rs1258000233	1012	dbSNP
rs1008519908	1017	dbSNP
rs1201071443	1023	dbSNP
rs777212858	1028	dbSNP
rs891354493	1032	dbSNP
rs7801	1037	dbSNP
rs9842	1038	dbSNP
rs1306234336	1039	dbSNP
rs1189107766	1041	dbSNP
rs1268972628	1044	dbSNP
rs1480491399	1048	dbSNP
rs1192363500	1049	dbSNP
rs999707875	1060	dbSNP
rs1215509273	1063	dbSNP
rs1467179358	1065	dbSNP
rs1171041862	1067	dbSNP
rs1342849486	1073	dbSNP
rs753587756	1088	dbSNP
rs1292030481	1091	dbSNP
rs1301696272	1105	dbSNP
rs1405753477	1119	dbSNP
rs1277689126	1121	dbSNP
rs904000907	1127	dbSNP
rs568877526	1130	dbSNP
rs144777532	1134	dbSNP
rs189681903	1137	dbSNP
rs1219762679	1138	dbSNP
rs756107317	1143	dbSNP
rs1267584004	1147	dbSNP
rs1490423527	1151	dbSNP
rs916622866	1155	dbSNP
rs1260485529	1175	dbSNP
rs865806287	1176	dbSNP
rs1187042413	1186	dbSNP
rs1412922633	1194	dbSNP
rs142515861	1195	dbSNP
rs1166714308	1197	dbSNP
rs1803706	1199	dbSNP
rs1385597802	1201	dbSNP
rs1425601590	1202	dbSNP
rs1297760650	1203	dbSNP
rs546738586	1205	dbSNP
rs528327523	1206	dbSNP
rs1401878930	1207	dbSNP
rs1289995712	1208	dbSNP
rs1379147373	1213	dbSNP
rs561142350	1214	dbSNP
rs1227076417	1215	dbSNP
rs1360598124	1217	dbSNP
rs907795514	1224	dbSNP
rs1489000231	1226	dbSNP
rs767562522	1233	dbSNP
rs1236361294	1249	dbSNP
rs951884003	1260	dbSNP
rs1183520620	1268	dbSNP
rs1243545068	1270	dbSNP
rs920393890	1272	dbSNP
rs1803707	1277	dbSNP
rs974497936	1283	dbSNP
rs1425426507	1284	dbSNP
rs1159979251	1285	dbSNP
rs964427211	1286	dbSNP
rs372144755	1296	dbSNP
rs1008967329	1303	dbSNP
rs1490071715	1315	dbSNP
rs1306660319	1316	dbSNP
rs1355468541	1318	dbSNP
rs761167868	1320	dbSNP
rs955705784	1330	dbSNP
rs113461500	1331	dbSNP
rs1309876460	1334	dbSNP
rs369139530	1338	dbSNP
rs1212157746	1340	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.