>PDZD4|NM_032512|3'UTR
   1 GCTGCCCGGGCGGGTACACGGCCCAGGCCCAGGGAACCCCCTGGGGCCCCGGCCCTCACTCTCCTATAGAGATTGTGTGT
  81 GTGTGTGTGTGCGCGCGCGCGTGCTCGCTGTGCGCACGCACACATCTCCTCGTCTGGGTGTGCGCACAGGGCTTTGTTAG
 161 CAGAGAGAAGCCCCTGAGGAGAAGGGACGCTTTTCTTCCTTCTGCCCAAGTAAAGTGACCATGCCAGTGGCCAGCACTGG
 241 GGGCACACCTGTGATGGGCACCCCTTCAGCTGTGCGTGTGCATTCCCCATCCCCCATGCTCTTGCGTGTGCTTGCACGTG
 321 CACGCACACACACACCCAGTGCTCTCTCCACCCGACCCGTGTACTTGCAGACAGGGAAGCTGAGCTGAAAGGAGCACAAG
 401 AGAGTGTCCGGCTTCGCTGCTGAGCGCGGCCTCTCCCCGCCGCTGCGCACTGCAGTTATTTGTAGACAAAGGCACCCCTG
 481 ATTTTTGTGGTTTTTCTCCCTTTCTGTGCTTGCCAATAGTTGTTTTGTTTTGTGGACCTGCCCTGGGGGCTGGCAGCTCC
 561 TTCAGGCAGCCTGGCAGAAGTGGAACTCCCCTCTCCACTGATGGCTGGGAAGGGAGTTGGGGAGGAAGAGTGGGAGGGAG
 641 GGCTGGGGATGGATGGGAGGGAAGGGGAGGGAAAGGTGGGAGGAATGGGAAGCAGCTGTTCTGGTGGCTTCCACTCTGAG
 721 GTGTGGGAACGGGGAGCCTCTGGGGGGCACCTGTTTCCCCAGTGACTCAGAGAGAGCAAGACTGCAGGAACGGAGGGGTC
 801 CTGCAGGGTGTCCACCAAGAAGTCACAGAGCCCGTTCTGCGCCCCACACCATCTGGAGCAGGGGCTTCTCTTCTGAGTGA
 881 ACTGAGGAAGGTGCTTAGATGGTGAGGGCTCTAGAAACCAAGCCCCATGAGCAGCAGTGGAAGACCTCGGGCCATGCGGC
 961 CTGGGGAGGACTTGGTGGCGATCCGCAACCTGGACCCCAGTGAGAGGCGGTGGGCTGACTGGGAAGGAGAGGCCCCCAAC
1041 CTCCTCAGGATTTGCACGTGTGAACTAGGCTGCCTGTGGGGTGCCCCTTAGGCTTGGAGAGCCCCAGATTGGAGGCAGAC
1121 AGACTGCACCACCCCTTCCCCCCCTGCATCTCAAGAATAAAGCAAGCTGCCTTTGTACTTGGTTGAAAAAAAAAAAAAAA
1201 AAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cuaCGGGACCGGACAGGGGCGu 5' || | ||||| ||||||| Target 5' tgaGCGC-GGCCTCTCCCCGCc 3'	421 - 441	166.00	-27.40
2	miRNA 3' cuACG-GGACCGGACAGGGGCGu 5' ||| || ||| ||||| | Target 5' acTGCACCACCCCT-TCCCCCCc 3'	1123 - 1144	123.00	-15.30
3	miRNA 3' cuaCGG-GACC------GGAC--AGGGGcgu 5' ||| |||| |||| ||||| Target 5' ggaGCCTCTGGGGGGCACCTGTTTCCCCagt 3'	733 - 763	119.00	-23.20

Mutant ID	Mutant Position	Mutant Source
rs781853495	1	dbSNP
rs375849010	4	dbSNP
rs782080476	11	dbSNP
rs372978867	17	dbSNP
rs782645559	19	dbSNP
rs782010694	21	dbSNP
rs782384042	24	dbSNP
rs368917754	29	dbSNP
rs1441492399	32	dbSNP
rs994894464	33	dbSNP
rs782487475	34	dbSNP
rs781971869	41	dbSNP
rs782196573	43	dbSNP
rs781842955	45	dbSNP
rs782564473	51	dbSNP
rs1178533355	57	dbSNP
rs1381525936	81	dbSNP
rs1441605141	82	dbSNP
rs1170958759	83	dbSNP
rs184981853	90	dbSNP
rs782529177	91	dbSNP
rs1334504960	92	dbSNP
rs1360657242	92	dbSNP
rs1444369782	92	dbSNP
rs781896560	92	dbSNP
rs781993143	92	dbSNP
rs940233866	92	dbSNP
rs1280347250	94	dbSNP
rs1456700823	99	dbSNP
rs908800330	100	dbSNP
rs1459482604	102	dbSNP
rs199802294	102	dbSNP
rs1478422245	105	dbSNP
rs113447867	106	dbSNP
rs939566904	111	dbSNP
rs1463865204	113	dbSNP
rs781968243	114	dbSNP
rs1393091460	116	dbSNP
rs980904613	117	dbSNP
rs1329970676	119	dbSNP
rs1436634328	128	dbSNP
rs1276236206	131	dbSNP
rs1402711268	132	dbSNP
rs530111872	132	dbSNP
rs781951950	142	dbSNP
rs183349961	144	dbSNP
rs1320556247	145	dbSNP
rs1208608158	149	dbSNP
rs915213791	151	dbSNP
rs1452243257	157	dbSNP
rs1201181740	167	dbSNP
rs1261220688	169	dbSNP
rs988120955	175	dbSNP
rs1477627352	179	dbSNP
rs1169637681	182	dbSNP
rs1424111985	184	dbSNP
rs1463369228	188	dbSNP
rs1161368501	194	dbSNP
rs782049123	198	dbSNP
rs956589189	199	dbSNP
rs575221741	221	dbSNP
rs1304433941	232	dbSNP
rs1337880826	239	dbSNP
rs1018851084	240	dbSNP
rs1399031920	249	dbSNP
rs1279189084	256	dbSNP
rs1009144023	261	dbSNP
rs1342753242	265	dbSNP
rs1243148789	271	dbSNP
rs953260823	272	dbSNP
rs1029145509	276	dbSNP
rs1211656722	278	dbSNP
rs1284003114	282	dbSNP
rs1488063979	291	dbSNP
rs1194619180	294	dbSNP
rs994566103	296	dbSNP
rs898931501	315	dbSNP
rs191831224	318	dbSNP
rs1430069201	320	dbSNP
rs1185226012	337	dbSNP
rs1367753386	342	dbSNP
rs1473091208	356	dbSNP
rs1004562338	359	dbSNP
rs782252457	363	dbSNP
rs1358104284	364	dbSNP
rs782296306	382	dbSNP
rs1298421374	385	dbSNP
rs1386774980	395	dbSNP
rs1394586601	397	dbSNP
rs939498744	399	dbSNP
rs1312481329	400	dbSNP
rs905390562	403	dbSNP
rs1341544630	410	dbSNP
rs1045654241	411	dbSNP
rs1248844370	412	dbSNP
rs1307769469	415	dbSNP
rs781986073	424	dbSNP
rs1217511577	425	dbSNP
rs915330006	426	dbSNP
rs1482401128	428	dbSNP
rs186992315	438	dbSNP
rs373555813	441	dbSNP
rs1424279456	442	dbSNP
rs911791046	446	dbSNP
rs1378471310	447	dbSNP
rs987433573	453	dbSNP
rs1438540748	465	dbSNP
rs953429719	485	dbSNP
rs1365126133	500	dbSNP
rs1464251587	504	dbSNP
rs1302595449	525	dbSNP
rs1403319570	528	dbSNP
rs1454962289	534	dbSNP
rs1329891604	538	dbSNP
rs1376897402	550	dbSNP
rs1240876635	554	dbSNP
rs1028800058	557	dbSNP
rs973644953	577	dbSNP
rs181912400	581	dbSNP
rs1461582649	583	dbSNP
rs1205405877	594	dbSNP
rs1236222199	598	dbSNP
rs963087476	604	dbSNP
rs782438581	608	dbSNP
rs1252089869	623	dbSNP
rs1430994257	631	dbSNP
rs1174888780	635	dbSNP
rs1377900608	639	dbSNP
rs1471158325	640	dbSNP
rs1168987005	644	dbSNP
rs1400133588	658	dbSNP
rs1448191707	661	dbSNP
rs782504773	663	dbSNP
rs1014718454	666	dbSNP
rs1386329874	669	dbSNP
rs1302244215	674	dbSNP
rs1325938550	676	dbSNP
rs1004550999	680	dbSNP
rs1255947685	706	dbSNP
rs895508580	718	dbSNP
rs1317184709	731	dbSNP
rs1205099673	736	dbSNP
rs1234579594	737	dbSNP
rs1487211797	741	dbSNP
rs1267530155	745	dbSNP
rs369643303	747	dbSNP
rs1481220414	750	dbSNP
rs1191892513	751	dbSNP
rs1423390973	777	dbSNP
rs1466503985	777	dbSNP
rs1173131257	779	dbSNP
rs1348702554	789	dbSNP
rs139252575	792	dbSNP
rs1294214272	795	dbSNP
rs1347567475	803	dbSNP
rs1003770077	804	dbSNP
rs782441503	808	dbSNP
rs1277850333	813	dbSNP
rs1339574280	815	dbSNP
rs1275022339	834	dbSNP
rs782666545	840	dbSNP
rs1220109851	841	dbSNP
rs375375896	845	dbSNP
rs946908447	849	dbSNP
rs1291897134	859	dbSNP
rs893889240	870	dbSNP
rs1213964539	878	dbSNP
rs1262076320	891	dbSNP
rs1475936340	892	dbSNP
rs1188666113	895	dbSNP
rs1412191995	899	dbSNP
rs1423769531	900	dbSNP
rs782498615	905	dbSNP
rs1165525145	915	dbSNP
rs782188044	948	dbSNP
rs1052494816	957	dbSNP
rs1361489238	979	dbSNP
rs935320400	980	dbSNP
rs1271727723	981	dbSNP
rs1376824977	984	dbSNP
rs911863078	985	dbSNP
rs987894012	1003	dbSNP
rs1315687968	1004	dbSNP
rs1360943686	1005	dbSNP
rs1213894056	1006	dbSNP
rs931876717	1007	dbSNP
rs781865965	1008	dbSNP
rs1236123378	1009	dbSNP
rs973255373	1010	dbSNP
rs1184729375	1018	dbSNP
rs1387696822	1023	dbSNP
rs1437902153	1025	dbSNP
rs1158129814	1033	dbSNP
rs1381087131	1038	dbSNP
rs1402684689	1040	dbSNP
rs1319995243	1058	dbSNP
rs1357178546	1067	dbSNP
rs2269367	1068	dbSNP
rs1014665865	1071	dbSNP
rs1337294687	1073	dbSNP
rs1384985846	1080	dbSNP
rs983229597	1082	dbSNP
rs959791314	1086	dbSNP
rs1035340217	1093	dbSNP
rs1225087691	1097	dbSNP
rs1001611843	1102	dbSNP
rs1311962480	1107	dbSNP
rs905410299	1117	dbSNP
rs1204786355	1119	dbSNP
rs1245439208	1125	dbSNP
rs1178043166	1129	dbSNP
rs1024347346	1134	dbSNP
rs1011011234	1138	dbSNP
rs894006002	1140	dbSNP
rs1469095735	1142	dbSNP
rs1444515539	1143	dbSNP
rs1330642896	1144	dbSNP
rs1335813525	1145	dbSNP
rs1443817485	1145	dbSNP
rs782472080	1145	dbSNP
rs782235210	1155	dbSNP
rs1343863304	1163	dbSNP
rs890537380	1178	dbSNP
rs782643819	1181	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.