>CPS1|NM_001122633|3'UTR
   1 AGATGCAGACACCCCAGCCCCATTATTAAATCAACCTGAGCCACATGTTATCTAAAGGAACTGATTCACAACTTTCTCAG
  81 AGATGAATATTGATAACTAAACTTCATTTCAGTTTACTTTGTTATGCCTTAATATTCTGTGTCTTTTGCAATTAAATTGT
 161 CAGTCACTTCTTCAAAACCTTACAGTCCTTCCTAAGTTACTCTTCATGAGATTTCATCCATTTACTAATACTGTATTTTT
 241 GGTGGACTAGGCTTGCCTATGTGCTTATGTGTAGCTTTTTACTTTTTATGGTGCTGATTAATGGTGATCAAGGTAGGAAA
 321 AGTTGCTGTTCTATTTTCTGAACTCTTTCTATACTTTAAGATACTCTATTTTTAAAACACTATCTGCAAACTCAGGACAC
 401 TTTAACAGGGCAGAATACTCTAAAAACTTGATAAAATTAAATATAGATTTAATTTATGAACCTTCCATCATGATGTTTGT
 481 GTATTGCTTCTTTTTGGATCCTCATTCTCACCCATTTGGCTAATCCAGGAATATTGTTATCCCTTCCCATTATATTGAAG
 561 TTGAGAAATGTGACAGAGGCATTTAGAGTATGGACTTTTCTTTTCTTTTTCTTTTTCTTTTTTTCTTTTTGAGATGGAGT
 641 CACACTCTCCAGGCTGGAGTGCAGTGGCACAATCTCGGCTCACTGCAATTTCCGTCTCCCAAGTTCAAGCGATTCTCCTG
 721 CTTTAGACTATGGATTTCTTTAAGGAATACTGGTTTGCAGTTTTGTTTTCTGGACTATATCAGCAGATGGTAGACAGTGT
 801 TTATGTAGATGTGTTGTTGTTTTTATCATTGGATTTTAACTTGGCCCGAGTGAAATAATCAGATTTTTGTCATTCACACT
 881 CTCCCCCAGTTTTGGAATAACTTGGAAGTAAGGTTCATTCCCTTAAGACGATGGATTCTGTTGAACTATGGGGTCCCACA
 961 CTGCACTATTAATTCCACCCACTGTAAGGGCAAGGACACCATTCCTTCTACATATAAGAAAAAAGTCTCTCCCCAAGGGC
1041 AGCCTTTGTTACTTTTAAATATTTTCTGTTATTACAAGTGCTCTAATTGTGAACTTTTAAATAAAATACTATTAAGAGGT
1121 AAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' agugUCAACG-GUCG-ACUCUAAu 5' |||| | | | ||||||| Target 5' cctaAGTTACTCTTCATGAGATTt 3'	191 - 214	150.00	-8.40
2	miRNA 3' agugucAACGGUCGACUCUAau 5' || |: :|||||| Target 5' tcttttTTTCTTTTTGAGATgg 3'	616 - 637	128.00	-6.40
3	miRNA 3' agUGUCAACGGUCGACUCUaau 5' |:| ||| ||:||||| Target 5' ttATA-TTG-AAGTTGAGAaat 3'	550 - 569	118.00	-10.92

Mutant ID	Mutant Position	Mutant Source
334037	6	ClinVar
334038	75	ClinVar
334039	188	ClinVar
334040	236	ClinVar
897793	275	ClinVar
334041	312	ClinVar
898935	315	ClinVar
334042	347	ClinVar
334043	381	ClinVar
334044	388	ClinVar
898936	425	ClinVar
898937	428	ClinVar
334045	439	ClinVar
898938	659	ClinVar
334046	693	ClinVar
334047	740	ClinVar
894806	748	ClinVar
334048	755	ClinVar
334049	778	ClinVar
894807	809	ClinVar
894808	830	ClinVar
894809	849	ClinVar
334050	850	ClinVar
896257	914	ClinVar
334051	947	ClinVar
334052	977	ClinVar
334053	1041	ClinVar
334054	1069	ClinVar
896258	1088	ClinVar
334055	1118	ClinVar
COSN26971749	3	COSMIC
COSN31500267	9	COSMIC
COSN31542136	14	COSMIC
COSN31500575	45	COSMIC
COSN31500044	51	COSMIC
COSN30181444	53	COSMIC
COSN30168160	93	COSMIC
COSN30509886	100	COSMIC
COSN30100182	107	COSMIC
COSN31501190	126	COSMIC
COSN9307246	185	COSMIC
COSN26491116	206	COSMIC
COSN5788141	242	COSMIC
COSN21631513	361	COSMIC
COSN29570957	573	COSMIC
COSN6166882	689	COSMIC
COSN5495990	1072	COSMIC
rs715	347	GWAS

Mutant ID	Mutant Position	Mutant Source
rs763770264	1	dbSNP
rs768304927	3	dbSNP
rs886055556	6	dbSNP
rs557721515	7	dbSNP
rs767908915	10	dbSNP
rs750819780	11	dbSNP
rs753480920	12	dbSNP
rs762783091	13	dbSNP
rs750331498	15	dbSNP
rs1171327996	17	dbSNP
rs766734493	18	dbSNP
rs373273939	30	dbSNP
rs1003072419	35	dbSNP
rs12475907	37	dbSNP
rs755007296	38	dbSNP
rs1457768473	45	dbSNP
rs765338172	46	dbSNP
rs757059771	50	dbSNP
rs752897106	51	dbSNP
rs781040449	52	dbSNP
rs745587710	53	dbSNP
rs1293254221	54	dbSNP
rs1192534859	58	dbSNP
rs898265774	62	dbSNP
rs755951811	63	dbSNP
rs868648409	65	dbSNP
rs886055557	75	dbSNP
rs780091328	84	dbSNP
rs1274036098	86	dbSNP
rs1013098567	94	dbSNP
rs768457837	94	dbSNP
rs749271521	98	dbSNP
rs768251900	100	dbSNP
rs774061608	103	dbSNP
rs796307701	128	dbSNP
rs996579632	133	dbSNP
rs1170715002	136	dbSNP
rs1411099676	137	dbSNP
rs1478447270	138	dbSNP
rs1181617197	151	dbSNP
rs1049451435	154	dbSNP
rs1015985104	158	dbSNP
rs577890646	166	dbSNP
rs890789032	167	dbSNP
rs1396051360	185	dbSNP
rs1412128096	187	dbSNP
rs543723956	188	dbSNP
rs1020873573	221	dbSNP
rs966996004	226	dbSNP
rs1360136890	232	dbSNP
rs6728169	236	dbSNP
rs954641404	241	dbSNP
rs766137873	246	dbSNP
rs1000668600	249	dbSNP
rs1032114219	252	dbSNP
rs959474286	260	dbSNP
rs1348574035	262	dbSNP
rs528584074	275	dbSNP
rs964012688	282	dbSNP
rs1366666132	288	dbSNP
rs556945268	289	dbSNP
rs1443967064	294	dbSNP
rs1352230512	307	dbSNP
rs917947121	308	dbSNP
rs886055558	312	dbSNP
rs115293884	315	dbSNP
rs983820997	322	dbSNP
rs1229830752	325	dbSNP
rs908129810	332	dbSNP
rs942234318	334	dbSNP
rs1431752459	336	dbSNP
rs1424941407	340	dbSNP
rs1177965565	342	dbSNP
rs1471307009	346	dbSNP
rs715	347	dbSNP
rs922081134	361	dbSNP
rs1486043070	369	dbSNP
rs947457050	370	dbSNP
rs200531041	380	dbSNP
rs10932349	381	dbSNP
rs1287602848	382	dbSNP
rs1307496743	383	dbSNP
rs886055559	388	dbSNP
rs1049489648	390	dbSNP
rs532362348	408	dbSNP
rs924769850	413	dbSNP
rs1287158273	414	dbSNP
rs1414802360	425	dbSNP
rs938960581	427	dbSNP
rs111328810	428	dbSNP
rs1465016073	428	dbSNP
rs943726342	434	dbSNP
rs1236247313	436	dbSNP
rs7684	439	dbSNP
rs902447837	443	dbSNP
rs1402662362	452	dbSNP
rs1409946050	466	dbSNP
rs1182837255	495	dbSNP
rs1379866668	496	dbSNP
rs1000720527	499	dbSNP
rs1259740792	505	dbSNP
rs1180017514	507	dbSNP
rs564483604	515	dbSNP
rs1032167035	525	dbSNP
rs1482915804	532	dbSNP
rs1261085113	543	dbSNP
rs1217768764	548	dbSNP
rs894976482	550	dbSNP
rs1267039961	554	dbSNP
rs1160504308	560	dbSNP
rs898951968	579	dbSNP
rs1356779157	582	dbSNP
rs1290954552	589	dbSNP
rs200158138	595	dbSNP
rs1379163098	601	dbSNP
rs561918056	601	dbSNP
rs796131504	601	dbSNP
rs993644143	601	dbSNP
rs1048028	606	dbSNP
rs1420089871	606	dbSNP
rs954507291	606	dbSNP
rs1162843033	612	dbSNP
rs1012327829	617	dbSNP
rs1188225745	618	dbSNP
rs550466354	618	dbSNP
rs1333361487	642	dbSNP
rs1017501207	644	dbSNP
rs1213903690	646	dbSNP
rs1449901166	647	dbSNP
rs964043790	649	dbSNP
rs559191307	659	dbSNP
rs971188679	670	dbSNP
rs1296861841	673	dbSNP
rs983487725	677	dbSNP
rs778492880	678	dbSNP
rs968859728	684	dbSNP
rs1350694920	685	dbSNP
rs1218134492	686	dbSNP
rs977889669	689	dbSNP
rs12476133	693	dbSNP
rs140637938	694	dbSNP
rs765822159	695	dbSNP
rs1204692026	698	dbSNP
rs916122180	699	dbSNP
rs565473415	710	dbSNP
rs111885056	711	dbSNP
rs1237998547	712	dbSNP
rs886055560	740	dbSNP
rs1456610013	752	dbSNP
rs1208895302	753	dbSNP
rs73078125	755	dbSNP
rs776991907	759	dbSNP
rs1252599489	761	dbSNP
rs1239029656	774	dbSNP
rs557541410	777	dbSNP
rs386654709	778	dbSNP
rs886055561	778	dbSNP
rs1340740639	780	dbSNP
rs371452070	804	dbSNP
rs943762320	809	dbSNP
rs560033629	812	dbSNP
rs1299410831	814	dbSNP
rs1416150601	815	dbSNP
rs929091141	819	dbSNP
rs79296773	830	dbSNP
rs536605142	833	dbSNP
rs181625711	835	dbSNP
rs1427736645	844	dbSNP
rs1369799935	847	dbSNP
rs573977144	848	dbSNP
rs142796725	849	dbSNP
rs150782174	850	dbSNP
rs1197687144	852	dbSNP
rs895030275	862	dbSNP
rs900450884	863	dbSNP
rs1012092578	866	dbSNP
rs572599114	879	dbSNP
rs1419356598	882	dbSNP
rs1011228031	883	dbSNP
rs1202499434	884	dbSNP
rs572914312	888	dbSNP
rs1274481683	889	dbSNP
rs906640205	903	dbSNP
rs1435067467	906	dbSNP
rs1232787626	908	dbSNP
rs541817831	914	dbSNP
rs1014959074	917	dbSNP
rs185214969	918	dbSNP
rs1290448013	925	dbSNP
rs963369822	927	dbSNP
rs973362412	930	dbSNP
rs1349973600	941	dbSNP
rs977488473	943	dbSNP
rs139770263	947	dbSNP
rs960199461	949	dbSNP
rs1171846087	962	dbSNP
rs1365116547	966	dbSNP
rs1179590622	968	dbSNP
rs556047307	974	dbSNP
rs763063376	977	dbSNP
rs563679146	981	dbSNP
rs1257646654	984	dbSNP
rs1189237692	992	dbSNP
rs916069853	998	dbSNP
rs529528829	1000	dbSNP
rs987676583	1001	dbSNP
rs764143400	1011	dbSNP
rs778290335	1026	dbSNP
rs1273922906	1030	dbSNP
rs886055562	1041	dbSNP
rs1351835942	1042	dbSNP
rs1270306960	1043	dbSNP
rs974043053	1044	dbSNP
rs1328619312	1047	dbSNP
rs965188425	1048	dbSNP
rs549195498	1057	dbSNP
rs1293932003	1063	dbSNP
rs920289554	1067	dbSNP
rs886055563	1069	dbSNP
rs1333422371	1078	dbSNP
rs1382625724	1084	dbSNP
rs760593059	1088	dbSNP
rs1156550306	1093	dbSNP
rs559545315	1101	dbSNP
rs1456338368	1109	dbSNP
rs1196813762	1111	dbSNP
rs1047516498	1113	dbSNP
rs528645400	1115	dbSNP
rs886055564	1118	dbSNP
rs1236295270	1119	dbSNP
rs1187434179	1120	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.