ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ccUCUUCUAACGUCUCAUUUAa 5' | ::|||| |:|||||| Target 5' ttATGGGATT--TGGGTAAATt 3'	452 - 471	140.00	-8.90
2	miRNA 3' ccucuUCUAACGUCUCAUUUAa 5' | ||| ||||||| | Target 5' ttctcACATT--AGAGTAATTt 3'	60 - 79	125.00	-6.32
3	miRNA 3' ccUCUUCUAACG-----UC-UCAUUUAa 5' | |||||| : || ||||| | Target 5' atACAAGATTATTATTGAGTAGTAACTg 3'	920 - 947	124.00	-6.59

Mutant ID	Mutant Position	Mutant Source
COSN30186019	6	COSMIC
COSN31565260	16	COSMIC
COSN31505307	33	COSMIC
COSN30476759	51	COSMIC
COSN31606839	63	COSMIC
COSN32065586	81	COSMIC
COSN28643728	83	COSMIC
COSN30530532	130	COSMIC
COSN19380288	135	COSMIC
COSN30111577	153	COSMIC
COSN24298707	158	COSMIC
COSN29802677	167	COSMIC
COSN15623270	173	COSMIC
COSN29728730	193	COSMIC
COSN28652925	195	COSMIC
COSN30131413	402	COSMIC
COSN31546302	473	COSMIC
COSN21778401	680	COSMIC
COSN1241111	761	COSMIC
COSN30120878	776	COSMIC
COSN30532002	793	COSMIC
COSN30124939	810	COSMIC
COSN17730821	874	COSMIC
COSN30545132	941	COSMIC
COSN31608838	992	COSMIC
COSN31517244	1007	COSMIC
COSN31534046	1086	COSMIC
COSN31530735	1122	COSMIC
COSN30530992	1285	COSMIC
COSN31538982	1297	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs146160188	1	dbSNP
rs768401002	6	dbSNP
rs1225397323	10	dbSNP
rs774186504	18	dbSNP
rs1283733563	22	dbSNP
rs762259985	24	dbSNP
rs570336122	25	dbSNP
rs138998287	26	dbSNP
rs1265676937	27	dbSNP
rs773794716	28	dbSNP
rs1191045871	32	dbSNP
rs756753426	35	dbSNP
rs761246811	35	dbSNP
rs766331465	42	dbSNP
rs1157407815	52	dbSNP
rs1032286853	56	dbSNP
rs1306545373	58	dbSNP
rs1377714912	63	dbSNP
rs541814813	64	dbSNP
rs892493686	65	dbSNP
rs1311790559	68	dbSNP
rs1009623533	79	dbSNP
rs1010695162	81	dbSNP
rs1304292542	82	dbSNP
rs3811616	83	dbSNP
rs971213945	103	dbSNP
rs1317359109	114	dbSNP
rs978013536	115	dbSNP
rs1362036182	119	dbSNP
rs6759249	121	dbSNP
rs187780923	135	dbSNP
rs577604070	145	dbSNP
rs544957050	149	dbSNP
rs1226168460	153	dbSNP
rs558107673	157	dbSNP
rs571842735	158	dbSNP
rs1282196130	172	dbSNP
rs975649876	173	dbSNP
rs958611053	178	dbSNP
rs141982185	179	dbSNP
rs3842562	182	dbSNP
rs201479976	184	dbSNP
rs931304740	184	dbSNP
rs770690918	187	dbSNP
rs542424382	188	dbSNP
rs1269569074	189	dbSNP
rs775513869	192	dbSNP
rs940097005	193	dbSNP
rs1136151	195	dbSNP
rs927779913	196	dbSNP
rs192525393	198	dbSNP
rs1055041158	203	dbSNP
rs769951436	208	dbSNP
rs893505894	209	dbSNP
rs915389126	211	dbSNP
rs1417964562	226	dbSNP
rs368359477	228	dbSNP
rs1010559458	231	dbSNP
rs1050549806	231	dbSNP
rs910743287	232	dbSNP
rs1485460726	236	dbSNP
rs1466825713	237	dbSNP
rs1238335800	240	dbSNP
rs1212295804	242	dbSNP
rs945143834	249	dbSNP
rs1305892471	254	dbSNP
rs1272410868	258	dbSNP
rs1219124753	259	dbSNP
rs1319922309	260	dbSNP
rs1172395323	262	dbSNP
rs1291194362	268	dbSNP
rs753452324	277	dbSNP
rs1379399012	280	dbSNP
rs1008616408	283	dbSNP
rs1042109593	284	dbSNP
rs1400259615	286	dbSNP
rs1384150847	297	dbSNP
rs1321287898	300	dbSNP
rs559763293	303	dbSNP
rs1163220351	304	dbSNP
rs771215608	318	dbSNP
rs1414777014	319	dbSNP
rs892441291	322	dbSNP
rs1009984245	326	dbSNP
rs1479800225	330	dbSNP
rs150823555	331	dbSNP
rs906604539	334	dbSNP
rs11897899	335	dbSNP
rs1271235668	342	dbSNP
rs1034168146	344	dbSNP
rs1322048209	345	dbSNP
rs185113508	346	dbSNP
rs1312726585	354	dbSNP
rs1221649313	357	dbSNP
rs1372174021	360	dbSNP
rs952736694	375	dbSNP
rs1448440107	380	dbSNP
rs958557355	384	dbSNP
rs1006114525	388	dbSNP
rs908522245	391	dbSNP
rs1197027551	396	dbSNP
rs1254043429	411	dbSNP
rs531270418	418	dbSNP
rs1016208468	421	dbSNP
rs1352375216	429	dbSNP
rs371596760	430	dbSNP
rs961698313	434	dbSNP
rs972130612	437	dbSNP
rs1251238061	444	dbSNP
rs923000915	459	dbSNP
rs1266928370	467	dbSNP
rs1488768985	467	dbSNP
rs1266901636	473	dbSNP
rs927822918	490	dbSNP
rs937848021	504	dbSNP
rs954593409	506	dbSNP
rs1055072776	508	dbSNP
rs1368088195	512	dbSNP
rs1302105001	516	dbSNP
rs1386641804	520	dbSNP
rs915181479	530	dbSNP
rs985871580	532	dbSNP
rs1295645056	539	dbSNP
rs1452133334	542	dbSNP
rs1194975811	551	dbSNP
rs910689114	554	dbSNP
rs936857862	556	dbSNP
rs1053922380	557	dbSNP
rs150012028	567	dbSNP
rs199542169	569	dbSNP
rs756766802	586	dbSNP
rs1160236451	588	dbSNP
rs1009100087	590	dbSNP
rs764724612	595	dbSNP
rs1485449788	604	dbSNP
rs1455664836	607	dbSNP
rs761826180	608	dbSNP
rs765396282	609	dbSNP
rs1046829749	614	dbSNP
rs190154708	615	dbSNP
rs192908054	618	dbSNP
rs1016049866	619	dbSNP
rs961394526	621	dbSNP
rs1381041573	623	dbSNP
rs1299193914	632	dbSNP
rs982635033	632	dbSNP
rs552811124	636	dbSNP
rs1002688541	645	dbSNP
rs1055228650	648	dbSNP
rs894290371	662	dbSNP
rs928053996	664	dbSNP
rs1466653622	679	dbSNP
rs571295337	682	dbSNP
rs1168172658	704	dbSNP
rs763280851	714	dbSNP
rs1263554871	716	dbSNP
rs1267038930	718	dbSNP
rs915046566	726	dbSNP
rs1256413512	731	dbSNP
rs1326670792	732	dbSNP
rs564695399	744	dbSNP
rs1346440343	747	dbSNP
rs946621520	761	dbSNP
rs1214945303	762	dbSNP
rs1016155740	771	dbSNP
rs543368346	772	dbSNP
rs977997691	773	dbSNP
rs77626683	776	dbSNP
rs923633791	777	dbSNP
rs944373533	779	dbSNP
rs144260379	794	dbSNP
rs993451314	800	dbSNP
rs1030397071	802	dbSNP
rs750869049	807	dbSNP
rs1296530726	808	dbSNP
rs954416102	809	dbSNP
rs1265670893	810	dbSNP
rs1473805471	811	dbSNP
rs931703974	813	dbSNP
rs1049892630	838	dbSNP
rs985818293	842	dbSNP
rs1187761650	849	dbSNP
rs888612578	852	dbSNP
rs1247731426	853	dbSNP
rs1183381387	859	dbSNP
rs1194789847	862	dbSNP
rs1457707788	873	dbSNP
rs13393608	874	dbSNP
rs1220829105	875	dbSNP
rs958164648	902	dbSNP
rs1284007925	908	dbSNP
rs1166983986	910	dbSNP
rs577189020	912	dbSNP
rs1245712713	913	dbSNP
rs1342502736	915	dbSNP
rs989599092	917	dbSNP
rs1350605738	921	dbSNP
rs1457315204	923	dbSNP
rs1446226144	926	dbSNP
rs1400912647	927	dbSNP
rs1333226005	931	dbSNP
rs897172310	935	dbSNP
rs1389659316	942	dbSNP
rs35089607	951	dbSNP
rs1163636258	954	dbSNP
rs1473566308	960	dbSNP
rs1361771468	964	dbSNP
rs1372352053	976	dbSNP
rs1177433739	977	dbSNP
rs1439826920	978	dbSNP
rs542561167	989	dbSNP
rs768204871	990	dbSNP
rs1035404971	1004	dbSNP
rs766849282	1023	dbSNP
rs990866614	1031	dbSNP
rs1210875731	1033	dbSNP
rs1358737844	1034	dbSNP
rs1348183578	1046	dbSNP
rs1278045518	1052	dbSNP
rs1022174790	1053	dbSNP
rs967863152	1079	dbSNP
rs150393218	1080	dbSNP
rs1438546634	1085	dbSNP
rs982416674	1090	dbSNP
rs978441957	1091	dbSNP
rs1406916638	1093	dbSNP
rs1312809977	1099	dbSNP
rs927927975	1101	dbSNP
rs1320636425	1107	dbSNP
rs1381558728	1108	dbSNP
rs758357319	1112	dbSNP
rs923861469	1112	dbSNP
rs1244673395	1113	dbSNP
rs1199992978	1123	dbSNP
rs536402558	1123	dbSNP
rs938012928	1123	dbSNP
rs1055175159	1125	dbSNP
rs1264155329	1125	dbSNP
rs11551407	1127	dbSNP
rs975785813	1128	dbSNP
rs1484341954	1129	dbSNP
rs1288236082	1135	dbSNP
rs1206240642	1145	dbSNP
rs888540800	1149	dbSNP
rs774845985	1158	dbSNP
rs1251620873	1159	dbSNP
rs941464687	1161	dbSNP
rs1438551793	1168	dbSNP
rs554421380	1170	dbSNP
rs931735243	1171	dbSNP
rs1400171314	1172	dbSNP
rs1048789871	1187	dbSNP
rs1171197688	1190	dbSNP
rs545299625	1192	dbSNP
rs1414593921	1193	dbSNP
rs1425059584	1193	dbSNP
rs1479290164	1196	dbSNP
rs139684271	1201	dbSNP
rs1037584731	1211	dbSNP
rs1391863589	1213	dbSNP
rs754622809	1215	dbSNP
rs184890970	1220	dbSNP
rs1279462697	1224	dbSNP
rs1030264758	1226	dbSNP
rs757704988	1227	dbSNP
rs73934339	1229	dbSNP
rs1449732328	1232	dbSNP
rs1303118217	1233	dbSNP
rs1378019074	1239	dbSNP
rs1341957673	1242	dbSNP
rs1310752247	1245	dbSNP
rs1007187185	1250	dbSNP
rs1226080606	1257	dbSNP
rs1017257761	1264	dbSNP
rs957781698	1266	dbSNP
rs34218417	1277	dbSNP
rs1299184158	1279	dbSNP
rs145198063	1290	dbSNP
rs1416639289	1291	dbSNP
rs1229868377	1297	dbSNP
rs1274327067	1313	dbSNP
rs1422187959	1316	dbSNP
rs1170370881	1318	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.