ID	Duplex structure	Position	Score	MFE
1	miRNA 3' guGGUCG-AUCGGAGUgg 5' ||:|| :|||||| Target 5' gcCCGGCACGGCCTCAgg 3'	3 - 20	131.00	-16.00
2	miRNA 3' guggUCGAUC-GGAGUGg 5' ||||:| |||||| Target 5' aaggAGCTGGCCCTCACa 3'	1235 - 1252	129.00	-17.30
3	miRNA 3' guggUCGA---UCGGAGUGg 5' |||| ||::|||| Target 5' gcagAGCTGGGAGTTTCACc 3'	899 - 918	121.00	-15.20

Mutant ID	Mutant Position	Mutant Source
rs763606269	2	dbSNP
rs1216992160	6	dbSNP
rs2230423	7	dbSNP
rs757059116	8	dbSNP
rs781189322	12	dbSNP
rs745795283	13	dbSNP
rs1475253904	15	dbSNP
rs1164585272	20	dbSNP
rs745471569	21	dbSNP
rs1305266674	22	dbSNP
rs768808444	26	dbSNP
rs1159525156	27	dbSNP
rs778933278	33	dbSNP
rs769366668	35	dbSNP
rs772415563	37	dbSNP
rs773636810	38	dbSNP
rs746235111	39	dbSNP
rs1277961022	40	dbSNP
rs1350185209	41	dbSNP
rs775252862	47	dbSNP
rs777218714	48	dbSNP
rs1266755786	49	dbSNP
rs762330996	52	dbSNP
rs1179730824	60	dbSNP
rs187874806	61	dbSNP
rs1308985733	62	dbSNP
rs1213125181	63	dbSNP
rs930246458	64	dbSNP
rs1352160655	70	dbSNP
rs1278223584	71	dbSNP
rs1205986518	85	dbSNP
rs192352004	93	dbSNP
rs948545265	94	dbSNP
rs1225291027	95	dbSNP
rs1293344162	102	dbSNP
rs1048675297	104	dbSNP
rs773851995	119	dbSNP
rs1295595699	121	dbSNP
rs1220141061	126	dbSNP
rs556015483	137	dbSNP
rs1001524055	139	dbSNP
rs1400986394	145	dbSNP
rs1055914079	146	dbSNP
rs1179098183	150	dbSNP
rs1456451645	154	dbSNP
rs1418516040	155	dbSNP
rs1189929202	158	dbSNP
rs768292892	163	dbSNP
rs1437766847	164	dbSNP
rs1193979301	167	dbSNP
rs183063780	169	dbSNP
rs1266400052	170	dbSNP
rs1208511402	172	dbSNP
rs898624832	173	dbSNP
rs1275637753	179	dbSNP
rs1227150273	181	dbSNP
rs1356238436	190	dbSNP
rs773241593	198	dbSNP
rs1283160370	208	dbSNP
rs903010718	219	dbSNP
rs1430734982	222	dbSNP
rs1366928790	223	dbSNP
rs1000100104	235	dbSNP
rs1434132825	238	dbSNP
rs777158947	242	dbSNP
rs1166797656	243	dbSNP
rs953071140	244	dbSNP
rs1412973817	245	dbSNP
rs1168572471	263	dbSNP
rs995552643	273	dbSNP
rs1321655578	277	dbSNP
rs985777432	292	dbSNP
rs1018996911	298	dbSNP
rs1188126564	299	dbSNP
rs759983838	300	dbSNP
rs3196658	305	dbSNP
rs1255341125	310	dbSNP
rs1321644584	319	dbSNP
rs972184429	320	dbSNP
rs1343628721	321	dbSNP
rs1356563256	323	dbSNP
rs1227275698	325	dbSNP
rs1371258794	341	dbSNP
rs41312134	353	dbSNP
rs1309003414	354	dbSNP
rs930120978	357	dbSNP
rs1349240400	359	dbSNP
rs1248837695	360	dbSNP
rs201242983	387	dbSNP
rs35418748	387	dbSNP
rs1431743994	394	dbSNP
rs1356230313	403	dbSNP
rs984402333	412	dbSNP
rs1287131841	413	dbSNP
rs1397046386	420	dbSNP
rs1189851220	432	dbSNP
rs1488329632	441	dbSNP
rs910118292	450	dbSNP
rs1008371051	451	dbSNP
rs753021343	453	dbSNP
rs933659472	456	dbSNP
rs1271929965	461	dbSNP
rs1203803155	479	dbSNP
rs1049972682	480	dbSNP
rs1198824062	481	dbSNP
rs1302881578	490	dbSNP
rs1217289611	499	dbSNP
rs1366600588	499	dbSNP
rs188235570	508	dbSNP
rs895772980	522	dbSNP
rs1339487395	527	dbSNP
rs1411642744	532	dbSNP
rs1456248805	533	dbSNP
rs572893880	536	dbSNP
rs1307502702	538	dbSNP
rs1435354533	543	dbSNP
rs1042183732	558	dbSNP
rs1804569	570	dbSNP
rs1174167324	578	dbSNP
rs1432788699	580	dbSNP
rs903042793	585	dbSNP
rs1185168515	587	dbSNP
rs924271232	594	dbSNP
rs956987207	598	dbSNP
rs1409238658	599	dbSNP
rs1179303724	607	dbSNP
rs1804568	609	dbSNP
rs751436746	626	dbSNP
rs1369036489	630	dbSNP
rs1471934307	644	dbSNP
rs1251646463	650	dbSNP
rs990312671	660	dbSNP
rs999966033	664	dbSNP
rs1257426177	678	dbSNP
rs1207639537	686	dbSNP
rs1032781996	696	dbSNP
rs1472423687	703	dbSNP
rs1161829091	704	dbSNP
rs1346004323	704	dbSNP
rs1455924621	704	dbSNP
rs772633678	704	dbSNP
rs867635378	704	dbSNP
rs1411906121	711	dbSNP
rs1161538166	718	dbSNP
rs1189219857	718	dbSNP
rs71947464	722	dbSNP
rs1421666246	723	dbSNP
rs915633347	725	dbSNP
rs1466355505	727	dbSNP
rs1330881847	728	dbSNP
rs889044149	729	dbSNP
rs200158453	730	dbSNP
rs1233876051	731	dbSNP
rs1310749748	731	dbSNP
rs1326762645	731	dbSNP
rs1395457945	731	dbSNP
rs1491378734	731	dbSNP
rs199636325	731	dbSNP
rs868274232	731	dbSNP
rs61387446	732	dbSNP
rs16990748	733	dbSNP
rs58007755	733	dbSNP
rs57536611	734	dbSNP
rs59751961	734	dbSNP
rs59133604	735	dbSNP
rs688969	735	dbSNP
rs1340474115	736	dbSNP
rs779563777	737	dbSNP
rs1281940797	739	dbSNP
rs981286230	742	dbSNP
rs928591028	743	dbSNP
rs971873349	748	dbSNP
rs1248290919	773	dbSNP
rs1026470394	776	dbSNP
rs1463249762	779	dbSNP
rs951549723	783	dbSNP
rs367956022	785	dbSNP
rs1218896698	788	dbSNP
rs1210209509	789	dbSNP
rs1257079322	797	dbSNP
rs1489425090	804	dbSNP
rs910023591	806	dbSNP
rs1244187785	829	dbSNP
rs747669043	840	dbSNP
rs937586121	846	dbSNP
rs1308114459	851	dbSNP
rs898512345	852	dbSNP
rs1364721593	856	dbSNP
rs931303890	856	dbSNP
rs749040540	857	dbSNP
rs1327078730	859	dbSNP
rs1205401522	860	dbSNP
rs768215054	877	dbSNP
rs992114501	882	dbSNP
rs771518919	884	dbSNP
rs1441782618	886	dbSNP
rs950078081	887	dbSNP
rs1056069	888	dbSNP
rs1056070	895	dbSNP
rs866091017	896	dbSNP
rs1390299162	902	dbSNP
rs774010970	909	dbSNP
rs1019770720	916	dbSNP
rs1389098258	918	dbSNP
rs903293995	928	dbSNP
rs1434638729	929	dbSNP
rs1265223415	933	dbSNP
rs1209208657	943	dbSNP
rs936058106	946	dbSNP
rs1170025126	947	dbSNP
rs1483329599	949	dbSNP
rs1276936447	956	dbSNP
rs999782113	970	dbSNP
rs1341732157	977	dbSNP
rs1032560687	983	dbSNP
rs1235925837	987	dbSNP
rs1471078237	1002	dbSNP
rs1173536015	1003	dbSNP
rs1054223791	1012	dbSNP
rs1401875318	1020	dbSNP
rs957037722	1022	dbSNP
rs1369583271	1024	dbSNP
rs1172761605	1025	dbSNP
rs199742261	1031	dbSNP
rs747472229	1037	dbSNP
rs989814578	1038	dbSNP
rs1341649793	1046	dbSNP
rs1197891079	1058	dbSNP
rs772737811	1070	dbSNP
rs1472917408	1073	dbSNP
rs888908844	1078	dbSNP
rs969826458	1086	dbSNP
rs1482744694	1090	dbSNP
rs760553708	1096	dbSNP
rs1277005317	1098	dbSNP
rs1343117176	1099	dbSNP
rs1007470200	1101	dbSNP
rs1255839825	1104	dbSNP
rs1040686936	1110	dbSNP
rs901538403	1112	dbSNP
rs771597894	1114	dbSNP
rs1286947304	1126	dbSNP
rs1407492288	1131	dbSNP
rs770866635	1134	dbSNP
rs939825619	1143	dbSNP
rs777324758	1144	dbSNP
rs919821093	1146	dbSNP
rs1171789329	1161	dbSNP
rs1457525732	1177	dbSNP
rs1156902171	1178	dbSNP
rs1025957678	1203	dbSNP
rs1049736492	1205	dbSNP
rs1286315157	1206	dbSNP
rs192955667	1210	dbSNP
rs1005932786	1211	dbSNP
rs1261043317	1212	dbSNP
rs1017120401	1225	dbSNP
rs1041110859	1241	dbSNP
rs1481551783	1248	dbSNP
rs1228374046	1258	dbSNP
rs1199793621	1264	dbSNP
rs184659322	1265	dbSNP
rs1464894783	1270	dbSNP
rs1160635540	1276	dbSNP
rs1361804387	1277	dbSNP
rs775505331	1298	dbSNP
rs763246013	1301	dbSNP
rs1435320470	1303	dbSNP
rs999665133	1315	dbSNP
rs1456700000	1328	dbSNP
rs1032612778	1340	dbSNP
rs894045844	1341	dbSNP
rs1471823276	1353	dbSNP
rs375146294	1354	dbSNP
rs15090	1359	dbSNP
rs917478696	1360	dbSNP
rs1189500204	1381	dbSNP
rs1466405633	1383	dbSNP
rs1246639112	1398	dbSNP
rs1388181371	1399	dbSNP
rs764109940	1401	dbSNP
rs1208491371	1402	dbSNP
rs971645832	1403	dbSNP
rs1301589451	1408	dbSNP
rs1229563980	1410	dbSNP
rs1353602008	1411	dbSNP
rs1313703359	1415	dbSNP
rs1228755976	1421	dbSNP
rs977845326	1427	dbSNP
rs1318297560	1434	dbSNP
rs751666385	1467	dbSNP
rs1022627251	1468	dbSNP
rs1230864420	1470	dbSNP
rs970088784	1474	dbSNP
rs1281064320	1476	dbSNP
rs1354337792	1477	dbSNP
rs1169287247	1482	dbSNP
rs981224361	1482	dbSNP
rs1215739131	1483	dbSNP
rs765483945	1487	dbSNP
rs1388482184	1496	dbSNP
rs1284004832	1497	dbSNP
rs961214642	1504	dbSNP
rs1249004023	1506	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.