>PNRC1|NM_006813|3'UTR
   1 ATTTCAGATTTCAGTATGTGTGTAAAACATAATTTTTCCCATATCCCTGGACTCTTGAGAAAATTGGTACAGAAATGGAA
  81 ATTTGCCTTGTTGCAACATACAATTGCAAAAGATGAGTTTAAAAAATTACATACAAACAGCTTGTATTATATTTTATATT
 161 TTGTAAATACTGTATACCATGTATTATGTGTATATTGTTCATACTTGAGAGGTATATTATAGTTTTGTTATGAAAGTATG
 241 TATTTTGCCCTGCCCACATTGCAGGTGTTTTGTATATATACAATGGATAAATTTTAAGTGTGTGCTAAGGCACATGGAAG
 321 ACCGATTTTATTTGCACAAGGTACTGAGATTTTTTTCAAGAAACAGCTGTCAAATCTCAAGGTGAAGATCTAAATGTGAA
 401 CAGTTTACTAATGCACTACTGAAGTTTAAATCTGTGGCACAATCAATGTAAGCATGGGGTTTGTTTCTCTAAATTGATTT
 481 GTAATCTGAAATTACTGAACAACTCCTATTCCCATTTTTGCTAAACTCAATTTCTGGTTTTGGTATATATCCATTCCAGC
 561 TTAATGCCTCTAATTTTAATGCCAACAAAATTGGTTGTAATCAAATTTTAAAATAATAATAATTTGGCCCCCCCTTTTAA
 641 AATAGTCTTGACTCTTTGTGTGTGACTGTTTCTCATGTTTGAATGTGTGACTAGGAGATGATTTTGTGTGGTTGGATTTT
 721 TTTGACTTCTACTTTACTGGCTGAGTGTGAGCCGCCATGCCTGGCCATAATCTACATTTTCTTACCAGGAGCAGCATTGA
 801 GGTTTTTGAGCATAGTACTTGACTACTCTAGAGGCTGAGACGGGAGCATCTCTTGAGCCTGAGAAGTGGAGATTGCAATT
 881 GAGCTAGGATCAGGCCACTGCACTCCAGCCTGGGTAACAGACGCTGTCTCAAAAAAAAGGCCAAGAGAAAGTAAGGGAGA
 961 CAGA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uacUAACGGAGGGAGAACUCUu 5' |||| :|| : ||||||| Target 5' tatATTG-TTCATACTTGAGAg 3'	191 - 211	158.00	-12.50
2	miRNA 3' uacUA-ACGGA-GGGAGAACUCUu 5' || | ||| ||||||||| Target 5' cccATATCCCTGGACTCTTGAGAa 3'	38 - 61	155.00	-17.20
3	miRNA 3' uacuaaCGGAGGGAGAACUCuu 5' || | |||||||| Target 5' acgggaGCAT--CTCTTGAGcc 3'	840 - 859	132.00	-14.50

Mutant ID	Mutant Position	Mutant Source
COSN9915647	33	COSMIC
COSN30479874	39	COSMIC
COSN30158404	73	COSMIC
COSN25155509	80	COSMIC
COSN23446634	104	COSMIC
COSN8514891	132	COSMIC
COSN31595764	171	COSMIC
COSN26596102	223	COSMIC
COSN31608636	291	COSMIC
COSN31563571	357	COSMIC
COSN31548227	433	COSMIC
COSN32018528	433	COSMIC
COSN28776900	487	COSMIC
COSN31585876	560	COSMIC
COSN6336309	683	COSMIC
COSN29619617	713	COSMIC
COSN23884749	716	COSMIC
COSN7902290	753	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs753601806	5	dbSNP
rs557616385	7	dbSNP
rs544879522	15	dbSNP
rs1262645385	17	dbSNP
rs1275050396	17	dbSNP
rs1262426369	19	dbSNP
rs1023509610	21	dbSNP
rs1429615017	27	dbSNP
rs1351687655	29	dbSNP
rs768759337	30	dbSNP
rs1266361955	36	dbSNP
rs776477624	37	dbSNP
rs779445312	40	dbSNP
rs747483218	44	dbSNP
rs1034955236	46	dbSNP
rs1400473076	47	dbSNP
rs769076378	53	dbSNP
rs79315464	65	dbSNP
rs988221689	71	dbSNP
rs1395795166	78	dbSNP
rs2231278	79	dbSNP
rs527623897	87	dbSNP
rs914957946	102	dbSNP
rs946497810	104	dbSNP
rs1375082884	105	dbSNP
rs542887135	106	dbSNP
rs1194336659	107	dbSNP
rs1180077899	108	dbSNP
rs1485589905	113	dbSNP
rs1255991546	114	dbSNP
rs1213700282	115	dbSNP
rs1305793170	121	dbSNP
rs975910598	121	dbSNP
rs570274584	122	dbSNP
rs1203931221	128	dbSNP
rs531223830	128	dbSNP
rs910743334	130	dbSNP
rs1219007485	133	dbSNP
rs543575735	136	dbSNP
rs112659597	141	dbSNP
rs1310945796	145	dbSNP
rs1416579698	145	dbSNP
rs1050790552	147	dbSNP
rs1182042571	150	dbSNP
rs914202317	150	dbSNP
rs1408343506	151	dbSNP
rs1398906785	152	dbSNP
rs1242276994	159	dbSNP
rs149150276	160	dbSNP
rs191825797	164	dbSNP
rs1416169333	165	dbSNP
rs1190643367	179	dbSNP
rs1417322992	180	dbSNP
rs183549807	182	dbSNP
rs1176157252	184	dbSNP
rs1360452051	188	dbSNP
rs1467887861	189	dbSNP
rs902935899	191	dbSNP
rs1002599791	192	dbSNP
rs1291216755	195	dbSNP
rs1235000868	196	dbSNP
rs1057218362	202	dbSNP
rs765965193	204	dbSNP
rs1049566417	206	dbSNP
rs1278960546	212	dbSNP
rs1440078983	215	dbSNP
rs887857759	217	dbSNP
rs188230479	219	dbSNP
rs1013037671	220	dbSNP
rs1461148062	221	dbSNP
rs1392648365	224	dbSNP
rs534250145	226	dbSNP
rs1013146629	236	dbSNP
rs143283530	240	dbSNP
rs529350149	251	dbSNP
rs1176025373	253	dbSNP
rs1468134419	257	dbSNP
rs1272375320	258	dbSNP
rs1004614197	264	dbSNP
rs1178796074	266	dbSNP
rs1467764512	269	dbSNP
rs1360498665	274	dbSNP
rs1215333812	275	dbSNP
rs1322151378	277	dbSNP
rs1003391235	278	dbSNP
rs1015642062	284	dbSNP
rs567924275	285	dbSNP
rs965435539	289	dbSNP
rs1279408168	297	dbSNP
rs1314069114	299	dbSNP
rs35617582	306	dbSNP
rs923076929	307	dbSNP
rs953336369	315	dbSNP
rs1328254411	316	dbSNP
rs1262785032	317	dbSNP
rs1437591725	319	dbSNP
rs956836931	345	dbSNP
rs1345019966	363	dbSNP
rs986031014	365	dbSNP
rs538250751	374	dbSNP
rs556550781	379	dbSNP
rs1195168557	383	dbSNP
rs1475038525	383	dbSNP
rs1488658799	399	dbSNP
rs542661009	402	dbSNP
rs1241334203	407	dbSNP
rs1213473294	413	dbSNP
rs1318667426	414	dbSNP
rs111668407	415	dbSNP
rs967804918	426	dbSNP
rs924306865	433	dbSNP
rs985818662	443	dbSNP
rs1387586436	446	dbSNP
rs181763524	447	dbSNP
rs556247486	449	dbSNP
rs1287503521	450	dbSNP
rs754358690	451	dbSNP
rs894605477	452	dbSNP
rs948909905	455	dbSNP
rs34654840	456	dbSNP
rs528483363	456	dbSNP
rs888693838	460	dbSNP
rs1004492141	467	dbSNP
rs1458036109	468	dbSNP
rs1418455466	470	dbSNP
rs1184542161	472	dbSNP
rs12445	482	dbSNP
rs919512614	487	dbSNP
rs776586550	503	dbSNP
rs929579041	505	dbSNP
rs1049048611	506	dbSNP
rs375604442	510	dbSNP
rs1234166275	514	dbSNP
rs948720194	515	dbSNP
rs1430617284	522	dbSNP
rs1276001778	530	dbSNP
rs187423047	537	dbSNP
rs779327505	541	dbSNP
rs1413508018	544	dbSNP
rs746265282	546	dbSNP
rs572227681	550	dbSNP
rs1021480291	552	dbSNP
rs1353552740	560	dbSNP
rs758713812	569	dbSNP
rs1478667834	571	dbSNP
rs977324234	586	dbSNP
rs1187086199	599	dbSNP
rs780188932	606	dbSNP
rs748063149	608	dbSNP
rs924259374	612	dbSNP
rs1258252053	618	dbSNP
rs1201104915	622	dbSNP
rs938354670	628	dbSNP
rs1249819100	629	dbSNP
rs1210890458	632	dbSNP
rs1200787543	633	dbSNP
rs1281864122	635	dbSNP
rs1293071582	637	dbSNP
rs571945117	655	dbSNP
rs992504805	655	dbSNP
rs1372043803	657	dbSNP
rs189796150	657	dbSNP
rs948538404	657	dbSNP
rs1056741329	658	dbSNP
rs1403319007	661	dbSNP
rs892375154	662	dbSNP
rs1009921018	669	dbSNP
rs888650676	671	dbSNP
rs1248410674	673	dbSNP
rs1400672016	675	dbSNP
rs1163932359	676	dbSNP
rs1022229783	677	dbSNP
rs1370087049	678	dbSNP
rs1188223811	680	dbSNP
rs940231946	683	dbSNP
rs1037289135	691	dbSNP
rs901138731	692	dbSNP
rs1322682870	693	dbSNP
rs967658684	707	dbSNP
rs1412154097	709	dbSNP
rs1272847548	713	dbSNP
rs1007187113	717	dbSNP
rs1358950678	721	dbSNP
rs1473360941	724	dbSNP
rs1017267695	725	dbSNP
rs889848958	729	dbSNP
rs1400521908	730	dbSNP
rs1377181398	731	dbSNP
rs1448428257	731	dbSNP
rs1011018036	732	dbSNP
rs1438415229	735	dbSNP
rs769863251	736	dbSNP
rs371181324	737	dbSNP
rs1021846084	738	dbSNP
rs1403106401	739	dbSNP
rs1302620640	741	dbSNP
rs1362078612	743	dbSNP
rs968542425	744	dbSNP
rs976212116	746	dbSNP
rs541845304	754	dbSNP
rs181875977	755	dbSNP
rs1268534283	757	dbSNP
rs1031559070	758	dbSNP
rs1349176556	761	dbSNP
rs1288869249	773	dbSNP
rs1241422390	777	dbSNP
rs1241647292	783	dbSNP
rs768689979	790	dbSNP
rs140613713	798	dbSNP
rs776760624	800	dbSNP
rs1230652306	804	dbSNP
rs959983879	813	dbSNP
rs748183863	819	dbSNP
rs1278037278	825	dbSNP
rs992452326	829	dbSNP
rs915604007	837	dbSNP
rs543062865	842	dbSNP
rs1228690790	843	dbSNP
rs1394579698	847	dbSNP
rs769879960	847	dbSNP
rs564891033	848	dbSNP
rs1171964193	849	dbSNP
rs78917993	853	dbSNP
rs983901358	855	dbSNP
rs186946679	859	dbSNP
rs565609339	860	dbSNP
rs774434071	864	dbSNP
rs1483774147	870	dbSNP
rs1037236545	874	dbSNP
rs766164164	876	dbSNP
rs773942046	879	dbSNP
rs1044001591	884	dbSNP
rs903781253	889	dbSNP
rs1164203211	893	dbSNP
rs1346673358	900	dbSNP
rs1275139740	902	dbSNP
rs1226376138	906	dbSNP
rs11760115	911	dbSNP
rs367981988	914	dbSNP
rs1423269751	915	dbSNP
rs1017256208	916	dbSNP
rs1050176261	919	dbSNP
rs767017808	919	dbSNP
rs76392013	923	dbSNP
rs1403144679	924	dbSNP
rs567962968	926	dbSNP
rs1167608401	928	dbSNP
rs775544335	930	dbSNP
rs1043822854	931	dbSNP
rs11427841	931	dbSNP
rs1474604292	931	dbSNP
rs531030757	931	dbSNP
rs536500597	931	dbSNP
rs1256098940	935	dbSNP
rs1327035101	940	dbSNP
rs998640481	942	dbSNP
rs1449120080	945	dbSNP
rs1278858756	946	dbSNP
rs1031506477	951	dbSNP
rs1315789315	955	dbSNP
rs755583022	959	dbSNP
rs1219104229	962	dbSNP
rs41273301	963	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.