>CIAPIN1|NM_020313|3'UTR
   1 GAGGTTCCTGACATGGGACCCATCTGCTCCTCCAGCCAACTCCTGTCCCTCACATCCCACCATGGTGGCTCCTCCCACCT
  81 CCTCTGGATTTGTTCACTCTGAGATCTGTTTGCAGAGTGGGTGCTTAGCAGACAGAGTGAAGCTGGCTGGGGGGCACAGT
 161 GGTGTGTAGTGCTGCTGTGTATCAAAAGACCAAGGTATTATGGGACCTGGTTTCAGAATGGGATGGGTTTCTTCACCTCA
 241 TGTTAAGAGAAGGGAGTGTGTCCTGAAGAAGCCCTTCTTCTGATGTTAAAATGCTGACCAGAACGCTCTTGAGCCCAGGC
 321 ATCGTTGAGCATTAACACTCTGTGACAGAGCTGCAGACCCCTGCCTTGAGTCTCATCTCAGCAATGCTGCCACCCTCTTG
 401 TCTTTCAGAGTTGTTAGTTTACTCCATTCTTTGTGACACGAGTCAAGTGGCTCACAACCTCCTCAGGGCACCAGAGGACT
 481 CACTCACTGGTTGCTGTGATGATATCCAGTGTCCCTCTGCCCCCTTCCATCCCCAACCACATTTGACTGTAGCATTGCAT
 561 CTGTGTCCTGTTGTCATTTATGTTAACCTTCAGGTATTAAACTTGCTGCATATCTTGACATATCTTGAGATTCTGCATGT
 641 CTTGTAAAGAGAGGGGATGTGCATTTGTGTGTGATGTTGGATAGTCATCCACGCTCAGTTTGGACCATTGGAGGAACTTA
 721 GTGTCACGCACAAATGGGGCTATTCCTACGCTTAGAATAGGGCTTGTCTGCCCACTTTAGAAGAGTCCAGGTTGGTGAGC
 801 ATTTAGAGGGAAGCAGGGCAGAACTCTGAACGACAATACGTCTCTCTGAGCAGAGACCCCTTTGTTCTTGTTATCCACCC
 881 ATATGGACTTGGAATCAATCTTGCCAAATATTTGGAGAGATTGTGTGGATTTAAGAGACCTGGATTTTTATATTTTACCA
 961 GTAAATAAAAGTTTTCATTGATATCTGTCCTTGAAACTTGAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' guGAGUCGCCGGCA--AAGGGGUu 5' ||| || || | |||||| Target 5' ccCTCTGCCCCCTTCCATCCCCAa 3'	513 - 536	126.00	-16.30
2	miRNA 3' guGAGUC--GCC---GGCAAAGGGGUu 5' :|||| :|| |||||::|| Target 5' gtTTCAGAATGGGATGGGTTTCTTCAc 3'	210 - 236	122.00	-13.00
3	miRNA 3' guGAGUCGC--CGG-CAAA-GGGGUu 5' :|||| | |:: |||| |:||| Target 5' ctTTCAGAGTTGTTAGTTTACTCCAt 3'	402 - 427	114.00	-12.70

Mutant ID	Mutant Position	Mutant Source
COSN30154892	26	COSMIC
COSN8601818	96	COSMIC
COSN1188323	181	COSMIC
COSN7261789	192	COSMIC
COSN1701220	663	COSMIC
COSN7261788	755	COSMIC
COSN195187	877	COSMIC
COSN22645470	983	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1453654571	3	dbSNP
rs1351137631	5	dbSNP
rs777129392	11	dbSNP
rs1361047175	16	dbSNP
rs370290851	40	dbSNP
rs747083763	42	dbSNP
rs778018151	43	dbSNP
rs1401354174	45	dbSNP
rs747957183	49	dbSNP
rs756334040	62	dbSNP
rs554370013	68	dbSNP
rs1202037717	73	dbSNP
rs1279048481	74	dbSNP
rs1294174596	79	dbSNP
rs1441497537	97	dbSNP
rs1185254222	102	dbSNP
rs1263551320	104	dbSNP
rs367916874	111	dbSNP
rs1253377640	112	dbSNP
rs536357279	115	dbSNP
rs569188131	117	dbSNP
rs1362607755	118	dbSNP
rs1468024599	121	dbSNP
rs750544408	122	dbSNP
rs1338835934	129	dbSNP
rs536042144	150	dbSNP
rs1310035664	151	dbSNP
rs140102313	152	dbSNP
rs553581852	153	dbSNP
rs1385612356	154	dbSNP
rs547843808	155	dbSNP
rs1160065873	166	dbSNP
rs1275222430	169	dbSNP
rs1346826162	170	dbSNP
rs1455481995	170	dbSNP
rs1410765191	171	dbSNP
rs1165274533	172	dbSNP
rs1475993303	174	dbSNP
rs1235177031	176	dbSNP
rs1208303424	178	dbSNP
rs767691099	181	dbSNP
rs1483160975	196	dbSNP
rs763193245	201	dbSNP
rs774746478	205	dbSNP
rs761906411	208	dbSNP
rs768989883	213	dbSNP
rs1464778629	217	dbSNP
rs1274026415	219	dbSNP
rs146238938	241	dbSNP
rs1429338014	246	dbSNP
rs774122300	257	dbSNP
rs1392886438	260	dbSNP
rs1354511186	268	dbSNP
rs764000279	274	dbSNP
rs1324068491	276	dbSNP
rs1283625869	294	dbSNP
rs1228308785	302	dbSNP
rs1231858299	304	dbSNP
rs1326921894	305	dbSNP
rs571218937	307	dbSNP
rs372263904	314	dbSNP
rs1322547081	315	dbSNP
rs867098526	317	dbSNP
rs1387567761	318	dbSNP
rs1326342720	321	dbSNP
rs141709574	323	dbSNP
rs1411207627	324	dbSNP
rs749689577	329	dbSNP
rs1391112590	331	dbSNP
rs538569544	336	dbSNP
rs1168547443	337	dbSNP
rs1370289599	340	dbSNP
rs1162100671	350	dbSNP
rs1458516572	354	dbSNP
rs1162706431	364	dbSNP
rs56116948	373	dbSNP
rs762679890	382	dbSNP
rs1475476974	384	dbSNP
rs1375277997	385	dbSNP
rs1365611602	396	dbSNP
rs1381324736	397	dbSNP
rs1293059159	401	dbSNP
rs1323117134	414	dbSNP
rs1187290061	418	dbSNP
rs1333358583	421	dbSNP
rs1211050124	426	dbSNP
rs567946716	428	dbSNP
rs188722211	429	dbSNP
rs1185116504	434	dbSNP
rs56150023	439	dbSNP
rs1424607608	440	dbSNP
rs1187382701	442	dbSNP
rs113582457	445	dbSNP
rs1289482571	456	dbSNP
rs1161223355	457	dbSNP
rs560639226	464	dbSNP
rs1389263207	468	dbSNP
rs1452841888	469	dbSNP
rs1286898882	471	dbSNP
rs1380966009	474	dbSNP
rs551543363	476	dbSNP
rs1292143283	478	dbSNP
rs368591924	505	dbSNP
rs373032693	506	dbSNP
rs1269373027	507	dbSNP
rs533378816	519	dbSNP
rs1316809089	522	dbSNP
rs184169089	523	dbSNP
rs1258119526	524	dbSNP
rs544178718	529	dbSNP
rs1438402853	536	dbSNP
rs1255521516	544	dbSNP
rs1473643244	549	dbSNP
rs1182951133	553	dbSNP
rs1347721766	559	dbSNP
rs1418421010	571	dbSNP
rs1322549331	587	dbSNP
rs1410023360	598	dbSNP
rs1466257377	601	dbSNP
rs1371804801	602	dbSNP
rs1311843091	604	dbSNP
rs1373214270	611	dbSNP
rs531533121	614	dbSNP
rs1449350348	621	dbSNP
rs1373318453	629	dbSNP
rs1396263384	637	dbSNP
rs1195859987	638	dbSNP
rs560839275	639	dbSNP
rs1246333938	651	dbSNP
rs1278510262	654	dbSNP
rs369567143	656	dbSNP
rs1484157681	667	dbSNP
rs1206034971	673	dbSNP
rs1182733434	674	dbSNP
rs377423246	680	dbSNP
rs547493888	681	dbSNP
rs1427031910	689	dbSNP
rs1454584571	692	dbSNP
rs1204557415	693	dbSNP
rs1341341069	696	dbSNP
rs1431239686	698	dbSNP
rs1281001281	703	dbSNP
rs1220730355	705	dbSNP
rs1328960513	707	dbSNP
rs148252667	708	dbSNP
rs1353347616	716	dbSNP
rs1365295864	725	dbSNP
rs1440688955	727	dbSNP
rs1274041589	728	dbSNP
rs1368879576	730	dbSNP
rs143160568	731	dbSNP
rs1301690281	734	dbSNP
rs776185885	736	dbSNP
rs770426982	739	dbSNP
rs1272421811	744	dbSNP
rs1436100180	747	dbSNP
rs748696018	749	dbSNP
rs1305586053	750	dbSNP
rs1192835789	761	dbSNP
rs1250842787	778	dbSNP
rs1450948617	779	dbSNP
rs1168094837	783	dbSNP
rs1418323268	783	dbSNP
rs1428588331	787	dbSNP
rs1166289009	790	dbSNP
rs544751685	791	dbSNP
rs1395929871	796	dbSNP
rs1440745130	805	dbSNP
rs1376591081	813	dbSNP
rs1178942619	815	dbSNP
rs1413281643	816	dbSNP
rs1324309615	818	dbSNP
rs1424823251	818	dbSNP
rs1367749197	822	dbSNP
rs1431784063	825	dbSNP
rs779307751	825	dbSNP
rs1159543662	826	dbSNP
rs1337655989	828	dbSNP
rs1473668803	828	dbSNP
rs1249955778	830	dbSNP
rs1230160998	831	dbSNP
rs149539447	831	dbSNP
rs556231118	832	dbSNP
rs1212916367	833	dbSNP
rs550723892	836	dbSNP
rs190531796	839	dbSNP
rs137898439	840	dbSNP
rs1310371875	842	dbSNP
rs1277604678	850	dbSNP
rs1474215562	853	dbSNP
rs1183112453	854	dbSNP
rs1417872292	855	dbSNP
rs1461410813	857	dbSNP
rs565409419	859	dbSNP
rs1391437404	860	dbSNP
rs1401687564	861	dbSNP
rs777268056	873	dbSNP
rs1318066188	876	dbSNP
rs1450394701	882	dbSNP
rs1224149378	883	dbSNP
rs555737739	892	dbSNP
rs534413663	893	dbSNP
rs1291409058	894	dbSNP
rs757875605	903	dbSNP
rs1286145816	906	dbSNP
rs749615599	908	dbSNP
rs1355478620	917	dbSNP
rs867673619	919	dbSNP
rs1444931033	922	dbSNP
rs55703233	923	dbSNP
rs549895246	924	dbSNP
rs1257634654	937	dbSNP
rs1474625032	943	dbSNP
rs1185112564	961	dbSNP
rs1446415694	962	dbSNP
rs1158364672	971	dbSNP
rs1359120130	971	dbSNP
rs1383168356	973	dbSNP
rs79500219	978	dbSNP
rs752631084	984	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.