>CTPS2|NM_001144002|3'UTR
   1 AATGAATACATGACTGGGAATAATGGGGACTGCCTGTGAGGCCTCTGAAATAATTGAAGGCAAGATGAAGGAACTATCTG
  81 AAGAAATCACTACACTCTTAGAGAATCCCTCTGTTCTCCAGCAAACATGGGATGTAAAGCCTCACAGGGAATCTGATAAT
 161 ACATACTTCTGTCAACCAGAACCAGAGGGGTAGTTTTCTTTTCCCTCCAGAGGCAGCCTTTGGTACTTAAAATATCTGTA
 241 GCTGATTAAATTTTTCCCAACAACCTCACTGGGGAGAAAGTGTGTTCATGTTTTGTCCAGCGGATCAGGATGTTAGGATG
 321 ACGAGCAAGAGTCCAGGTCACTGTGCCTTTGCTGTGTTGTATGGAAAGGATGGCAGGGAACATGCTGTAAGTAATTTTGA
 401 GTAAGAAAATGAGTCACTGTGTTACCTGGAACTCAGCCACAGATTTGTGTGTGGTCCAAGATCATTGCAGTTTCTCACCC
 481 TGTTTATTTCCTGGTAAAAGTAAAATTGAATAGGTCCAAGACTTGGGGGTGGCAAGTAAGGCTTTGCCTCAGGCACAAAA
 561 TTTAAGGGGGCTCCAAAAAACTCAGGAATCAAGATCAGCAATACAGTCTGAGTATCCCTTATGTGAAATGCTTGGGGCTA
 641 GAAGTGTTTTGAATTTCAGATTTTGGAATATTTGCATATACATGCGATATCTTGGGGATGAGACTCAAGACTAAACATGA
 721 AATTCATTTATGCTTCATATACACCTTATATACATAGCCTAAAGGTAATTTGATACAATATTTTAAATAATTTTGTGCAT
 801 GAAACAAAGTTTCGACTGCATTTTGACTGTGATTTCTGGCATGAGATCAGTTATGGAATTTTCCACTTCTAGCGTCATGT
 881 TGGCATTCAGAAATTTTGAAATTTTGGAGCATTTTGGATTTTCAGATTAGGGATGCTCAACCTGTATATATATTTTTTAA
 961 TCGACGTGAAATTCACGTAACATAGAATTAACCATTTTGAAGTGAACAATTTGGTTGCATTCACTGATGTTGAGCAACCA
1041 CCACCTTTAACTATTTCCAAAACATTTTCATCACTCCAAAATAAATGCCTGTACACACTAGCAGTCACTCCCTATCTTCC
1121 CCTCCACCTGTCCGCTGGCAACCACTGATCTCCTTTTTATTTCTGTGGCTTTTTCTATTCTGGATATTTCATATAAGTGG
1201 AATTACACAATATATGTGGTCTTTTGTGTCTGGCTTCTTCTGAGACAGTAGGAAGGGGGCTTGGCTTTGGCTCACCCCCA
1281 CTAGAGCATTTTTTCATGCATTCCCACTGATCACAAAACCCATACTACTACCTCATTGACACCATACCTGCTAACCTCGA
1361 GGCTTTAGTCATACAAAGAAAATGGCCTTTCTGTATTGTTCTTCTGTGCTCTCATAATGCTTAACCATGTCTTTTACTTA
1441 AACAATTCCAGGAACTGGCCTTAGGAGATCCAAATAGGGAACCAAGATTGCAGAGTGTCCCATCTTGGGAGGGAATGCTG
1521 AATAATTAATTGATTTACAGCCTTGTTGCCGCTGGCCAGACCACCAGGTGGCCCATTACTCGAGATGATCATCACAACCA
1601 GATGATGCTAACCTATATCCTCTACCCTTCGCGTGCTTTGTCTGGGAAGTCTTTTGGCCCCATGTCAGTTTCTATTGCAT
1681 TGAGAGCCCAAGAGCCCCTGGTCAGTCAGGCTTCCATTTAGCATGGCGTTTGCAAGGTTTACCCATGTTGTAGCATGTGT
1761 CAGAATTTCATTCCTTTCTATGGCTGAATAAAATTCCATTGTATGAATATACCACAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ggAGAUCAUU-UAUACAGUCGUa 5' | | |||: ||:|||||| | Target 5' caTGTTGTAGCATGTGTCAGAAt 3'	1744 - 1766	136.00	-12.52
2	miRNA 3' ggAGAUCAUUUA-UACAGUCGUa 5' ||||| :| ||||::||| Target 5' ctTCTAG--CGTCATGTTGGCAt 3'	866 - 886	128.00	-15.10
3	miRNA 3' ggaGA-UCAUUUAUAC-AGUCGUa 5' || || || | | |||||| Target 5' aaaCTCAGGAATCAAGATCAGCAa 3'	578 - 601	123.00	-9.23

Mutant ID	Mutant Position	Mutant Source
rs761978749	14	dbSNP
rs1396925091	18	dbSNP
rs1163314501	24	dbSNP
rs769328039	26	dbSNP
rs1424716104	28	dbSNP
rs776554892	36	dbSNP
rs1427221264	38	dbSNP
rs1260462795	41	dbSNP
rs769042421	46	dbSNP
rs1469586517	47	dbSNP
rs1045702729	48	dbSNP
rs183325683	57	dbSNP
rs780971074	58	dbSNP
rs111279848	65	dbSNP
rs950111604	74	dbSNP
rs751461755	78	dbSNP
rs1447051652	81	dbSNP
rs1191911661	96	dbSNP
rs1372994165	103	dbSNP
rs1448275830	109	dbSNP
rs1048854777	111	dbSNP
rs1527805	123	dbSNP
rs756774962	124	dbSNP
rs1386261142	125	dbSNP
rs1424200968	127	dbSNP
rs1292034777	130	dbSNP
rs756770372	131	dbSNP
rs1387461709	153	dbSNP
rs373691605	157	dbSNP
rs1416168984	172	dbSNP
rs115907493	181	dbSNP
rs911093022	184	dbSNP
rs1477589058	185	dbSNP
rs1315419149	191	dbSNP
rs1358508156	194	dbSNP
rs1264205714	206	dbSNP
rs1262101748	207	dbSNP
rs73448212	209	dbSNP
rs1204033412	217	dbSNP
rs955255343	231	dbSNP
rs190021009	238	dbSNP
rs916123999	239	dbSNP
rs185307190	246	dbSNP
rs764980871	249	dbSNP
rs1255846597	250	dbSNP
rs73448210	252	dbSNP
rs111774126	254	dbSNP
rs936269744	254	dbSNP
rs956728156	254	dbSNP
rs149224095	255	dbSNP
rs1454560214	256	dbSNP
rs766426217	257	dbSNP
rs1000991087	262	dbSNP
rs904016007	275	dbSNP
rs1023765784	276	dbSNP
rs976407366	282	dbSNP
rs964058155	293	dbSNP
rs1394056355	307	dbSNP
rs1340678654	333	dbSNP
rs1012428458	334	dbSNP
rs896712315	366	dbSNP
rs1335913813	371	dbSNP
rs1237893409	375	dbSNP
rs760843010	379	dbSNP
rs1265641185	382	dbSNP
rs1325111432	386	dbSNP
rs1203798219	387	dbSNP
rs191009457	394	dbSNP
rs929611920	397	dbSNP
rs896848657	399	dbSNP
rs1180472939	410	dbSNP
rs888387592	411	dbSNP
rs1157516932	413	dbSNP
rs1181402729	413	dbSNP
rs1038522055	415	dbSNP
rs1303252290	420	dbSNP
rs941160258	427	dbSNP
rs1410089532	437	dbSNP
rs1470986347	444	dbSNP
rs1025418734	447	dbSNP
rs113974347	449	dbSNP
rs1398049464	450	dbSNP
rs530005578	457	dbSNP
rs774751940	494	dbSNP
rs1336388485	525	dbSNP
rs6632849	557	dbSNP
rs6632848	561	dbSNP
rs768955929	563	dbSNP
rs1172803581	567	dbSNP
rs764481034	567	dbSNP
rs1312052719	568	dbSNP
rs1422331383	568	dbSNP
rs760886445	569	dbSNP
rs63711961	572	dbSNP
rs867261922	572	dbSNP
rs869064959	572	dbSNP
rs1182829564	574	dbSNP
rs775863048	575	dbSNP
rs1249878928	579	dbSNP
rs940641435	589	dbSNP
rs1200153302	596	dbSNP
rs1427741712	596	dbSNP
rs745669015	596	dbSNP
rs770753792	596	dbSNP
rs1172483227	603	dbSNP
rs1236891541	607	dbSNP
rs988716008	644	dbSNP
rs956696209	648	dbSNP
rs1046233851	654	dbSNP
rs1031039639	657	dbSNP
rs1014220645	664	dbSNP
rs144490864	669	dbSNP
rs775732355	670	dbSNP
rs1301926094	674	dbSNP
rs1364920568	691	dbSNP
rs1215860684	693	dbSNP
rs894456880	694	dbSNP
rs1308667179	695	dbSNP
rs1024152810	705	dbSNP
rs936227272	724	dbSNP
rs1012397531	726	dbSNP
rs926208380	739	dbSNP
rs1242122659	748	dbSNP
rs767876243	750	dbSNP
rs1313096692	757	dbSNP
rs1041886361	758	dbSNP
rs1434868804	767	dbSNP
rs1188407039	770	dbSNP
rs896679954	774	dbSNP
rs746543235	778	dbSNP
rs1035259261	779	dbSNP
rs994457640	787	dbSNP
rs1448854764	806	dbSNP
rs1447515018	825	dbSNP
rs1397268787	874	dbSNP
rs1424844809	878	dbSNP
rs1300024047	879	dbSNP
rs1347811430	880	dbSNP
rs777311446	881	dbSNP
rs1432277859	883	dbSNP
rs896813502	914	dbSNP
rs759992862	917	dbSNP
rs1363190168	920	dbSNP
rs1456539497	937	dbSNP
rs774880290	939	dbSNP
rs1165850937	942	dbSNP
rs1446837594	943	dbSNP
rs1378914487	968	dbSNP
rs889517401	974	dbSNP
rs1178563124	983	dbSNP
rs1320203967	1010	dbSNP
rs1049489141	1011	dbSNP
rs1458637530	1029	dbSNP
rs771680219	1030	dbSNP
rs1242777847	1034	dbSNP
rs1252019610	1036	dbSNP
rs1464339520	1048	dbSNP
rs1186617019	1052	dbSNP
rs140679622	1068	dbSNP
rs1444999195	1069	dbSNP
rs1162497505	1077	dbSNP
rs922471490	1078	dbSNP
rs1487929268	1082	dbSNP
rs373693699	1094	dbSNP
rs1265665858	1116	dbSNP
rs976901824	1129	dbSNP
rs1052962097	1136	dbSNP
rs1159124962	1138	dbSNP
rs1387305581	1161	dbSNP
rs761052109	1162	dbSNP
rs963868158	1183	dbSNP
rs910870654	1187	dbSNP
rs369789635	1205	dbSNP
rs925869948	1226	dbSNP
rs12010843	1233	dbSNP
rs968471192	1247	dbSNP
rs1284043984	1260	dbSNP
rs773512952	1268	dbSNP
rs1240762800	1278	dbSNP
rs1284983892	1284	dbSNP
rs1328618034	1288	dbSNP
rs1206962378	1298	dbSNP
rs771404273	1299	dbSNP
rs952602586	1302	dbSNP
rs1461768334	1309	dbSNP
rs1186500781	1319	dbSNP
rs1025533290	1326	dbSNP
rs56111615	1327	dbSNP
rs1470931944	1329	dbSNP
rs916652397	1335	dbSNP
rs990932377	1337	dbSNP
rs1410823113	1345	dbSNP
rs377292018	1352	dbSNP
rs1178038007	1353	dbSNP
rs993975216	1355	dbSNP
rs1309188741	1357	dbSNP
rs1415546974	1362	dbSNP
rs1385161181	1363	dbSNP
rs187023858	1369	dbSNP
rs994341780	1374	dbSNP
rs748855029	1382	dbSNP
rs1444584068	1385	dbSNP
rs780174709	1386	dbSNP
rs1350167470	1397	dbSNP
rs1426863845	1423	dbSNP
rs961618699	1429	dbSNP
rs756215885	1444	dbSNP
rs1324112506	1449	dbSNP
rs1199826252	1458	dbSNP
rs1276196829	1459	dbSNP
rs1439310044	1468	dbSNP
rs1205016864	1481	dbSNP
rs772703014	1483	dbSNP
rs1017621307	1489	dbSNP
rs1250594967	1493	dbSNP
rs750511891	1502	dbSNP
rs1358987478	1507	dbSNP
rs1024842239	1518	dbSNP
rs769334192	1525	dbSNP
rs142245573	1528	dbSNP
rs1366085362	1546	dbSNP
rs780868462	1549	dbSNP
rs1172534134	1566	dbSNP
rs894585036	1574	dbSNP
rs1055777576	1581	dbSNP
rs1049459019	1593	dbSNP
rs754758278	1594	dbSNP
rs1000216293	1598	dbSNP
rs868340981	1607	dbSNP
rs1370117371	1608	dbSNP
rs1408248141	1661	dbSNP
rs1304985360	1689	dbSNP
rs1331789010	1701	dbSNP
rs904739037	1718	dbSNP
rs1042000782	1734	dbSNP
rs1341454771	1746	dbSNP
rs1214934344	1759	dbSNP
rs946354485	1765	dbSNP
rs922759557	1768	dbSNP
rs1267523081	1782	dbSNP
rs1224277249	1785	dbSNP
rs373128413	1789	dbSNP
rs780201166	1790	dbSNP
rs1052850168	1794	dbSNP
rs1217108034	1797	dbSNP
rs1352669923	1802	dbSNP
rs1127358	1813	dbSNP
rs1169557787	1815	dbSNP
rs762126793	1829	dbSNP
rs925869364	1833	dbSNP
rs1391791677	1835	dbSNP
rs1330590980	1849	dbSNP
rs73630553	1850	dbSNP
rs1328056589	1874	dbSNP
rs946726369	1882	dbSNP
rs1435738427	1893	dbSNP
rs973052572	1897	dbSNP
rs758409116	1918	dbSNP
rs990901261	1930	dbSNP
rs960744243	1938	dbSNP
rs1247949388	1941	dbSNP
rs753974293	1942	dbSNP
rs1292019188	1951	dbSNP
rs1338741223	1952	dbSNP
rs1207487155	1953	dbSNP
rs1297925604	1958	dbSNP
rs1263236992	1969	dbSNP
rs1449188683	1985	dbSNP
rs1189814126	1995	dbSNP
rs1247998560	2004	dbSNP
rs1448024477	2005	dbSNP
rs1185875427	2009	dbSNP
rs1401652808	2010	dbSNP
rs751739023	2013	dbSNP
rs1457595549	2027	dbSNP
rs1161473020	2067	dbSNP
rs1387257083	2075	dbSNP
rs113105334	2087	dbSNP
rs763254840	2109	dbSNP
rs972869290	2138	dbSNP
rs961864702	2144	dbSNP
rs1025208139	2150	dbSNP
rs1017170544	2157	dbSNP
rs1313168267	2178	dbSNP
rs1012097356	2184	dbSNP
rs756314821	2188	dbSNP
rs776013888	2201	dbSNP
rs188843365	2202	dbSNP
rs200481825	2231	dbSNP
rs867979021	2231	dbSNP
rs200968778	2237	dbSNP
rs398087652	2237	dbSNP
rs74432602	2237	dbSNP
rs200148636	2238	dbSNP
rs1207615133	2250	dbSNP
rs1463985493	2252	dbSNP
rs1205894085	2256	dbSNP
rs1257313359	2259	dbSNP
rs1423410685	2266	dbSNP
rs1185726991	2269	dbSNP
rs772704911	2272	dbSNP
rs1027906098	2286	dbSNP
rs997813769	2289	dbSNP
rs900901558	2299	dbSNP
rs771628852	2304	dbSNP
rs142757741	2306	dbSNP
rs767604370	2312	dbSNP
rs759993937	2319	dbSNP
rs1386283017	2321	dbSNP
rs1470924796	2322	dbSNP
rs1303165465	2333	dbSNP
rs1448506714	2335	dbSNP
rs1401271195	2352	dbSNP
rs774684350	2355	dbSNP
rs1276328324	2361	dbSNP
rs1308670602	2368	dbSNP
rs766936521	2370	dbSNP
rs1199773389	2383	dbSNP
rs1345691518	2392	dbSNP
rs763383304	2401	dbSNP
rs1263779924	2403	dbSNP
rs73630552	2419	dbSNP
rs1256911453	2422	dbSNP
rs111751185	2425	dbSNP
rs1041580733	2434	dbSNP
rs1254227316	2444	dbSNP
rs1277816662	2449	dbSNP
rs1483508306	2467	dbSNP
rs948685419	2475	dbSNP
rs181847051	2479	dbSNP
rs1481354624	2480	dbSNP
rs1332668168	2487	dbSNP
rs1176669150	2497	dbSNP
rs1377053832	2505	dbSNP
rs781109635	2517	dbSNP
rs912900198	2519	dbSNP
rs1055098242	2527	dbSNP
rs939348039	2528	dbSNP
rs928013577	2529	dbSNP
rs1408849897	2541	dbSNP
rs988510051	2559	dbSNP
rs973255516	2586	dbSNP
rs749062171	2598	dbSNP
rs1219346320	2602	dbSNP
rs188332238	2607	dbSNP
rs889492902	2608	dbSNP
rs910079293	2609	dbSNP
rs984307130	2612	dbSNP
rs1343080168	2617	dbSNP
rs59001637	2624	dbSNP
rs16656	2626	dbSNP
rs772289010	2626	dbSNP
rs1048138748	2628	dbSNP
rs1489495421	2640	dbSNP
rs1219	2645	dbSNP
rs1028544401	2651	dbSNP
rs776383263	2665	dbSNP
rs1476211869	2671	dbSNP
rs1390580693	2672	dbSNP
rs143725184	2684	dbSNP
rs757285209	2702	dbSNP
rs746871638	2704	dbSNP
rs184119500	2705	dbSNP
rs1292502173	2709	dbSNP
rs1367449091	2719	dbSNP
rs56279107	2730	dbSNP
rs192945552	2736	dbSNP
rs999045950	2737	dbSNP
rs1244894749	2738	dbSNP
rs1205572756	2751	dbSNP
rs904354888	2756	dbSNP
rs1272743031	2770	dbSNP
rs1021839456	2772	dbSNP
rs1323804691	2781	dbSNP
rs779931113	2784	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.