>GPRIN1|NM_052899|3'UTR
   1 TCTGCCCCCATTTTGTACGCCCGAGTTTCCGACCTTCTCAGGCTCCCTTCTTGATCACAGGCCCCTAGAAGGGGTCCCCT
  81 CTGCGTGCCACAGGCCTCCGAGGGGTTGTGCAGCCTCTAGGGCTGTTGCGTCCCCGTCTTTCCAGCCCCTCCCATCACAA
 161 ATACAGAAGAACCCCTTCTACCAAGCTCCCTCGTGGCCACGAGGCCACGAACCCGGCCAGCCCTGACGCCCCACTGTCCC
 241 CTCACAGCCCTCAGCTCTACTCCCGGTCACACTGGGCGACCACGGGGGCCTGCTCAGCACCCTCACCTCCCACCCCCTGA
 321 GAGCTGGGGCAGGCTCCTGAGATGTCTAGACTGGTGCGTTGTGGTCTCCGGTGGGGCCAGATCCCCAGAACAGGGAGAGA
 401 CTGCATACAAGTCTGAGTGGCAGAAGCTTCAAGTGGGTGAGGATCGGTGTGTGAGACCCGCGAGTGGGCACGTCTCTGAA
 481 TGTGAGGTCTCGGGCAGCACGTGAGGTAGAACGAGTGTGAGCGTCTCCACGCAGTGGCTGGCCCCGTGGCCGGGGCGTCT
 561 CTTGCATGCTGTTGGTGGTCCTTCCCCAGTTCCTATCTCCCACCCGCCTGGTTCCAGCCCCATCCCCTCTCCCACTGAGT
 641 CACAGGTTTGAGATTCCCAGAGAGGCCAAAGACAGCTACAGGAAGTACCCTGGGGGCTCAGCTGAGAGAGAGTCACAGAA
 721 AGGCCAGAGGGTCTCAGGAAGGTCTGGAGGTCACAGCTGGGCTCACAGAAGCTCTTGGGCCCTGGCATCCTGGGTGACCG
 801 GTGTTCTTGGGTATTGGGGACAGGACAGAGAGCTCATTCTATAACCACAGTTGTCTTTTAAAGGCCACCCTCTCCAGCCC
 881 CTTGTCCCCCCTGTGCTGTGAGCCCAAGTCCCTTGTGACCACTCAGTGTCCCGTCCCCACCCCTCCAGCCGCAGTTTTTG
 961 GCTACAAACTGTCACAGTATACATTGGTAATAAAATATTTCCCCAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gcGGCC--CGAGGGGCCCc 5' | || |: |||:||| Target 5' taCAGGAAGTACCCTGGGg 3'	677 - 695	129.00	-18.60
2	miRNA 3' gcGGCCCGAGGGGCCCc 5' || ||| :||:||| Target 5' gcCCTGGCATCCTGGGt 3'	779 - 795	127.00	-19.10
3	miRNA 3' gcGGCCCG-AGGGGCCCc 5' |:|||| || |||| Target 5' caCTGGGCGACCACGGGg 3'	270 - 287	123.00	-27.50

Mutant ID	Mutant Position	Mutant Source
COSN30672587	10	COSMIC
COSN31556038	18	COSMIC
COSN30466279	23	COSMIC
COSN30462254	30	COSMIC
COSN30475689	46	COSMIC
COSN28228748	53	COSMIC
COSN31533490	65	COSMIC
COSN31563608	99	COSMIC
COSN30605077	129	COSMIC
COSN31597295	149	COSMIC
COSN1312431	640	COSMIC
COSN19463524	756	COSMIC
COSN30478982	1002	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1198217393	1	dbSNP
rs1004810356	3	dbSNP
rs748639847	4	dbSNP
rs372449779	5	dbSNP
rs567990984	7	dbSNP
rs368776103	12	dbSNP
rs1352231583	15	dbSNP
rs751697057	22	dbSNP
rs780252128	23	dbSNP
rs372334299	24	dbSNP
rs750624829	30	dbSNP
rs1013658129	32	dbSNP
rs1328142561	35	dbSNP
rs1263563051	36	dbSNP
rs1446170712	40	dbSNP
rs763904167	43	dbSNP
rs760422330	53	dbSNP
rs1468711586	55	dbSNP
rs752652611	62	dbSNP
rs1416052174	64	dbSNP
rs767615426	65	dbSNP
rs930688043	68	dbSNP
rs1490330686	69	dbSNP
rs894933642	73	dbSNP
rs527963695	74	dbSNP
rs759099986	78	dbSNP
rs774069772	79	dbSNP
rs1360562651	84	dbSNP
rs1313035281	85	dbSNP
rs770565506	87	dbSNP
rs1410108889	89	dbSNP
rs1309468426	91	dbSNP
rs762605559	92	dbSNP
rs1434773487	97	dbSNP
rs1430807047	100	dbSNP
rs1274575678	107	dbSNP
rs772760828	116	dbSNP
rs1346364251	119	dbSNP
rs1221404864	135	dbSNP
rs1272632418	136	dbSNP
rs1341527193	140	dbSNP
rs1206222433	145	dbSNP
rs962298242	149	dbSNP
rs28572012	159	dbSNP
rs112164239	162	dbSNP
rs1450028209	171	dbSNP
rs1197932257	174	dbSNP
rs1247030859	190	dbSNP
rs530803258	192	dbSNP
rs1186574609	195	dbSNP
rs969943572	200	dbSNP
rs1023414051	204	dbSNP
rs1011085803	208	dbSNP
rs1363351214	209	dbSNP
rs1404347643	211	dbSNP
rs570058962	212	dbSNP
rs945665212	217	dbSNP
rs1332018285	223	dbSNP
rs957787405	228	dbSNP
rs912785458	230	dbSNP
rs1296037550	235	dbSNP
rs11541045	238	dbSNP
rs1365022306	240	dbSNP
rs1335771541	241	dbSNP
rs1276804946	242	dbSNP
rs1051129901	247	dbSNP
rs1209438259	249	dbSNP
rs932634885	254	dbSNP
rs1030626884	261	dbSNP
rs747047528	263	dbSNP
rs1247355387	264	dbSNP
rs559699419	265	dbSNP
rs1396663599	270	dbSNP
rs901130284	272	dbSNP
rs1040936328	281	dbSNP
rs533380321	284	dbSNP
rs962753459	288	dbSNP
rs1021419938	291	dbSNP
rs983672003	293	dbSNP
rs559861068	301	dbSNP
rs780227662	313	dbSNP
rs541618783	315	dbSNP
rs930728453	318	dbSNP
rs959035775	319	dbSNP
rs1436410770	320	dbSNP
rs920645736	327	dbSNP
rs1338758668	334	dbSNP
rs1244415084	336	dbSNP
rs6862187	343	dbSNP
rs940671788	345	dbSNP
rs1000586514	347	dbSNP
rs903591533	351	dbSNP
rs1465318331	358	dbSNP
rs1210488815	361	dbSNP
rs1021144859	363	dbSNP
rs1233482700	365	dbSNP
rs562361928	368	dbSNP
rs1198276448	370	dbSNP
rs1157443208	374	dbSNP
rs1411650659	380	dbSNP
rs1458276805	388	dbSNP
rs543453041	390	dbSNP
rs1400245101	395	dbSNP
rs891409806	396	dbSNP
rs1334858730	400	dbSNP
rs1342514507	401	dbSNP
rs1226761505	406	dbSNP
rs1255482420	412	dbSNP
rs1051779753	421	dbSNP
rs1329587804	439	dbSNP
rs982759223	446	dbSNP
rs1254179450	460	dbSNP
rs575946755	468	dbSNP
rs770148360	469	dbSNP
rs530147231	471	dbSNP
rs115870095	483	dbSNP
rs1225043065	491	dbSNP
rs1481892619	492	dbSNP
rs143413319	500	dbSNP
rs1273711701	506	dbSNP
rs1376452846	509	dbSNP
rs1030700314	512	dbSNP
rs999186219	513	dbSNP
rs775687630	515	dbSNP
rs1373093309	517	dbSNP
rs1377328520	523	dbSNP
rs1326566981	524	dbSNP
rs1408297143	530	dbSNP
rs941444123	531	dbSNP
rs1019516875	535	dbSNP
rs553578945	545	dbSNP
rs1007230490	546	dbSNP
rs889684305	548	dbSNP
rs568408789	551	dbSNP
rs1161064402	552	dbSNP
rs1257298564	557	dbSNP
rs535266411	561	dbSNP
rs1048713412	569	dbSNP
rs58939354	571	dbSNP
rs899288981	577	dbSNP
rs1483800901	580	dbSNP
rs1179348150	581	dbSNP
rs1452727775	589	dbSNP
rs1265665491	599	dbSNP
rs992495646	604	dbSNP
rs1036431621	606	dbSNP
rs555997707	609	dbSNP
rs1397038689	621	dbSNP
rs959255845	622	dbSNP
rs1033326439	629	dbSNP
rs979046198	642	dbSNP
rs868275459	658	dbSNP
rs537052105	664	dbSNP
rs940702794	665	dbSNP
rs1194744955	667	dbSNP
rs1021113620	675	dbSNP
rs1464843092	682	dbSNP
rs570003394	683	dbSNP
rs1009775947	686	dbSNP
rs1227049710	689	dbSNP
rs906546273	697	dbSNP
rs1297638880	704	dbSNP
rs1264110029	713	dbSNP
rs551597699	713	dbSNP
rs780526273	713	dbSNP
rs114992997	716	dbSNP
rs1210741814	722	dbSNP
rs1255455609	723	dbSNP
rs566177392	723	dbSNP
rs77768830	727	dbSNP
rs1188041998	729	dbSNP
rs1447187309	742	dbSNP
rs1430570291	771	dbSNP
rs916804343	776	dbSNP
rs1413730047	778	dbSNP
rs941334581	781	dbSNP
rs990088845	782	dbSNP
rs887239311	789	dbSNP
rs149638893	792	dbSNP
rs1394248131	794	dbSNP
rs1297583788	798	dbSNP
rs950130792	800	dbSNP
rs923714415	807	dbSNP
rs1359805041	809	dbSNP
rs536646294	811	dbSNP
rs61198452	811	dbSNP
rs746094782	811	dbSNP
rs965068609	821	dbSNP
rs1311574567	836	dbSNP
rs1019714750	839	dbSNP
rs985112080	841	dbSNP
rs1275396703	846	dbSNP
rs529470194	855	dbSNP
rs1208627784	865	dbSNP
rs954135031	878	dbSNP
rs1346003408	882	dbSNP
rs967683397	884	dbSNP
rs768002048	886	dbSNP
rs1486889087	887	dbSNP
rs116373545	893	dbSNP
rs1180124195	893	dbSNP
rs778309422	894	dbSNP
rs995772151	896	dbSNP
rs896978489	897	dbSNP
rs1409454416	898	dbSNP
rs1456692246	902	dbSNP
rs550468153	903	dbSNP
rs1346825891	904	dbSNP
rs1445191482	909	dbSNP
rs1301699778	913	dbSNP
rs1015408059	915	dbSNP
rs1379481939	923	dbSNP
rs1004989497	929	dbSNP
rs906577104	932	dbSNP
rs1396892519	933	dbSNP
rs1285584788	935	dbSNP
rs1319194638	936	dbSNP
rs1208143131	937	dbSNP
rs1030261328	938	dbSNP
rs1174370279	939	dbSNP
rs1190308589	947	dbSNP
rs138301358	951	dbSNP
rs111390019	953	dbSNP
rs1418041928	955	dbSNP
rs948071449	971	dbSNP
rs1412646321	972	dbSNP
rs1359214791	973	dbSNP
rs564142639	977	dbSNP
rs1005769330	980	dbSNP
rs1237283135	983	dbSNP
rs1409617991	986	dbSNP
rs1054021715	991	dbSNP
rs936869187	1001	dbSNP
rs924123633	1006	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.