ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ccGGAGGCCUUCGAGGCGGGGu 5' ||||| || ||||||| Target 5' gtCCTCC----GC-CCGCCCCc 3'	31 - 47	153.00	-25.50
2	miRNA 3' ccGGAGGC--CUUCGAGGCGGGGu 5' ||| || | || |||:||| Target 5' ccCCTGCGCTGTTGCCCCGTCCCc 3'	804 - 827	142.00	-22.50
3	miRNA 3' ccggaggcCUUCGAGGCGGGGu 5' ||||| |||:||| Target 5' tgcagggaGAAGC-CCGTCCCc 3'	534 - 554	141.00	-21.00

Mutant ID	Mutant Position	Mutant Source
COSN24314863	3	COSMIC
COSN24314864	4	COSMIC
COSN13844702	25	COSMIC
COSN18742324	33	COSMIC
COSN18742325	34	COSMIC
COSN30481058	37	COSMIC
COSN31542899	38	COSMIC
COSN26991251	40	COSMIC
COSN8553233	44	COSMIC
COSN30119514	48	COSMIC
COSN26991249	49	COSMIC
COSN30190482	50	COSMIC
COSN30158032	62	COSMIC
COSN30524835	84	COSMIC
COSN30506225	100	COSMIC
COSN2531324	105	COSMIC
COSN1219990	128	COSMIC
COSN1219991	371	COSMIC
COSN5762667	423	COSMIC
COSN32058370	579	COSMIC
rs2241588	44	GWAS

Mutant ID	Mutant Position	Mutant Source
rs1349891533	2	dbSNP
rs370720760	4	dbSNP
rs373539999	8	dbSNP
rs200780966	9	dbSNP
rs1437834579	10	dbSNP
rs11556322	12	dbSNP
rs773886522	14	dbSNP
rs1426119549	21	dbSNP
rs763311062	22	dbSNP
rs751029054	25	dbSNP
rs569556993	26	dbSNP
rs752531404	29	dbSNP
rs755939071	31	dbSNP
rs370209262	32	dbSNP
rs538572417	33	dbSNP
rs548496605	34	dbSNP
rs375840799	38	dbSNP
rs374172999	39	dbSNP
rs1260996319	41	dbSNP
rs781532025	42	dbSNP
rs748341027	43	dbSNP
rs2241588	44	dbSNP
rs377340131	48	dbSNP
rs199953755	49	dbSNP
rs373537972	53	dbSNP
rs1331689528	62	dbSNP
rs1465428935	67	dbSNP
rs1398472901	77	dbSNP
rs878954258	78	dbSNP
rs920428883	80	dbSNP
rs191106406	91	dbSNP
rs950403489	92	dbSNP
rs981778880	94	dbSNP
rs1189529971	96	dbSNP
rs1200410139	97	dbSNP
rs1239019568	100	dbSNP
rs1045174039	105	dbSNP
rs905228332	108	dbSNP
rs1249356815	110	dbSNP
rs556177076	114	dbSNP
rs937761016	121	dbSNP
rs1300568582	122	dbSNP
rs762615637	124	dbSNP
rs1378562464	125	dbSNP
rs566019031	128	dbSNP
rs111822635	129	dbSNP
rs946898461	133	dbSNP
rs542109178	134	dbSNP
rs1174653541	136	dbSNP
rs763776687	137	dbSNP
rs904001724	140	dbSNP
rs1178131749	155	dbSNP
rs1190502863	164	dbSNP
rs1042637835	167	dbSNP
rs1183360387	168	dbSNP
rs1478045286	170	dbSNP
rs1483489530	173	dbSNP
rs1000392208	177	dbSNP
rs1355786498	193	dbSNP
rs902773322	196	dbSNP
rs182133113	197	dbSNP
rs1314500525	199	dbSNP
rs1421432628	200	dbSNP
rs1343965533	205	dbSNP
rs1298654620	208	dbSNP
rs1378810216	211	dbSNP
rs572221238	212	dbSNP
rs1051065840	219	dbSNP
rs751046318	220	dbSNP
rs1361774843	223	dbSNP
rs962300230	226	dbSNP
rs1325721946	228	dbSNP
rs1006918607	232	dbSNP
rs1017459626	233	dbSNP
rs1301321407	237	dbSNP
rs1356548799	240	dbSNP
rs898997650	241	dbSNP
rs1185570209	243	dbSNP
rs1489212152	244	dbSNP
rs998011555	245	dbSNP
rs1285768625	246	dbSNP
rs1215241016	247	dbSNP
rs866362173	250	dbSNP
rs1485067317	252	dbSNP
rs541100232	254	dbSNP
rs1026194909	255	dbSNP
rs111863045	258	dbSNP
rs950693125	262	dbSNP
rs981749171	266	dbSNP
rs1034696147	267	dbSNP
rs1451796029	271	dbSNP
rs959055280	275	dbSNP
rs1192529321	276	dbSNP
rs1341660475	281	dbSNP
rs564148282	282	dbSNP
rs1250595691	283	dbSNP
rs939352724	291	dbSNP
rs1392891353	295	dbSNP
rs533095940	296	dbSNP
rs915014666	299	dbSNP
rs1416010040	300	dbSNP
rs373442658	302	dbSNP
rs1164751822	303	dbSNP
rs563321281	305	dbSNP
rs924194667	306	dbSNP
rs1445221251	309	dbSNP
rs1268776592	310	dbSNP
rs1209940646	312	dbSNP
rs1440437713	313	dbSNP
rs185331184	318	dbSNP
rs1270941615	319	dbSNP
rs1379133549	320	dbSNP
rs1448194758	327	dbSNP
rs945643036	328	dbSNP
rs1051318297	329	dbSNP
rs548875261	341	dbSNP
rs1333355781	342	dbSNP
rs1247503696	348	dbSNP
rs568530219	353	dbSNP
rs1356696555	354	dbSNP
rs1038448370	356	dbSNP
rs1466339473	357	dbSNP
rs1270747003	358	dbSNP
rs6603066	359	dbSNP
rs1179212390	363	dbSNP
rs994639637	365	dbSNP
rs1048039515	366	dbSNP
rs1249782305	366	dbSNP
rs1379345052	367	dbSNP
rs1198931296	371	dbSNP
rs1417215449	372	dbSNP
rs542609577	373	dbSNP
rs1457946425	374	dbSNP
rs1008590576	376	dbSNP
rs1019052470	381	dbSNP
rs886326745	383	dbSNP
rs1368757675	384	dbSNP
rs1003447287	386	dbSNP
rs1034958447	387	dbSNP
rs961736840	389	dbSNP
rs547602565	390	dbSNP
rs972427291	392	dbSNP
rs550504397	402	dbSNP
rs570574162	402	dbSNP
rs1402581555	403	dbSNP
rs1404255505	405	dbSNP
rs1022005751	407	dbSNP
rs556911079	408	dbSNP
rs939444393	408	dbSNP
rs1439882242	413	dbSNP
rs1232001620	421	dbSNP
rs570074472	422	dbSNP
rs1444559051	428	dbSNP
rs1257305031	430	dbSNP
rs1210719422	436	dbSNP
rs1346366650	437	dbSNP
rs924093626	439	dbSNP
rs1241010705	440	dbSNP
rs955564158	441	dbSNP
rs1371878327	443	dbSNP
rs987012451	452	dbSNP
rs913929948	453	dbSNP
rs1341976066	455	dbSNP
rs1334582948	456	dbSNP
rs911511228	457	dbSNP
rs1223098218	458	dbSNP
rs942667712	459	dbSNP
rs535371620	461	dbSNP
rs919996999	462	dbSNP
rs1008643023	463	dbSNP
rs1319522504	467	dbSNP
rs1187713852	469	dbSNP
rs930062854	470	dbSNP
rs1484957048	471	dbSNP
rs555662737	472	dbSNP
rs886254668	473	dbSNP
rs993139620	474	dbSNP
rs1028003548	480	dbSNP
rs1003415814	481	dbSNP
rs1256622406	483	dbSNP
rs112488926	484	dbSNP
rs895067082	488	dbSNP
rs1011892870	489	dbSNP
rs1344032523	492	dbSNP
rs534735739	497	dbSNP
rs1406697748	502	dbSNP
rs1414114603	503	dbSNP
rs1164627176	507	dbSNP
rs1474466421	509	dbSNP
rs1022392147	515	dbSNP
rs1185488253	517	dbSNP
rs371101635	518	dbSNP
rs967767253	520	dbSNP
rs1478262151	521	dbSNP
rs1223909777	529	dbSNP
rs1452373858	531	dbSNP
rs1272842449	532	dbSNP
rs1033535954	549	dbSNP
rs1332321979	550	dbSNP
rs1279871528	551	dbSNP
rs138277625	551	dbSNP
rs575421223	551	dbSNP
rs771511502	551	dbSNP
rs960525420	554	dbSNP
rs992278158	554	dbSNP
rs999662678	554	dbSNP
rs1030746127	556	dbSNP
rs913951685	557	dbSNP
rs1303890831	558	dbSNP
rs979431525	558	dbSNP
rs1375257766	559	dbSNP
rs1252455058	563	dbSNP
rs925537723	563	dbSNP
rs1191918721	565	dbSNP
rs1280468027	566	dbSNP
rs1342053161	568	dbSNP
rs1049965412	571	dbSNP
rs932930186	571	dbSNP
rs1306821651	572	dbSNP
rs1214723072	573	dbSNP
rs12985124	574	dbSNP
rs912767225	576	dbSNP
rs1294245128	578	dbSNP
rs11556318	579	dbSNP
rs1305207595	580	dbSNP
rs944179273	584	dbSNP
rs12986016	586	dbSNP
rs955528755	587	dbSNP
rs1299072976	588	dbSNP
rs1461839716	592	dbSNP
rs1353257734	593	dbSNP
rs1235366007	594	dbSNP
rs1280999604	595	dbSNP
rs541192371	598	dbSNP
rs12985145	602	dbSNP
rs1208531800	604	dbSNP
rs1239064245	605	dbSNP
rs993583859	607	dbSNP
rs12985152	611	dbSNP
rs12985163	618	dbSNP
rs191223269	622	dbSNP
rs1488744260	625	dbSNP
rs12985169	628	dbSNP
rs543587080	630	dbSNP
rs1417492323	631	dbSNP
rs1434945186	641	dbSNP
rs1355376908	655	dbSNP
rs12985203	660	dbSNP
rs1307338769	662	dbSNP
rs12983454	668	dbSNP
rs1391174970	669	dbSNP
rs563125248	671	dbSNP
rs12985400	677	dbSNP
rs919910384	679	dbSNP
rs930018815	684	dbSNP
rs1375410953	697	dbSNP
rs983270229	698	dbSNP
rs1421657400	704	dbSNP
rs1381143874	709	dbSNP
rs1158574976	713	dbSNP
rs1013381485	725	dbSNP
rs1021474802	733	dbSNP
rs1300508726	742	dbSNP
rs528914231	743	dbSNP
rs1361505018	748	dbSNP
rs966789106	763	dbSNP
rs879002301	766	dbSNP
rs1287885059	767	dbSNP
rs542132772	768	dbSNP
rs925327030	774	dbSNP
rs1220498444	775	dbSNP
rs1260815435	778	dbSNP
rs1489024565	778	dbSNP
rs939128587	783	dbSNP
rs954039438	784	dbSNP
rs545854786	787	dbSNP
rs985684484	787	dbSNP
rs1347142393	791	dbSNP
rs1311661348	792	dbSNP
rs562374940	793	dbSNP
rs912775842	794	dbSNP
rs1317243380	795	dbSNP
rs1385691048	798	dbSNP
rs1288391016	802	dbSNP
rs1056330095	807	dbSNP
rs182942495	808	dbSNP
rs1166800292	809	dbSNP
rs895034454	811	dbSNP
rs972899648	812	dbSNP
rs1185627144	818	dbSNP
rs1478774427	821	dbSNP
rs547539773	822	dbSNP
rs947951977	823	dbSNP
rs1266895982	825	dbSNP
rs1453128382	826	dbSNP
rs1043671110	828	dbSNP
rs903512635	829	dbSNP
rs1160922022	831	dbSNP
rs1381352301	835	dbSNP
rs999232651	836	dbSNP
rs777323263	837	dbSNP
rs1030672493	840	dbSNP
rs887474723	841	dbSNP
rs937623713	849	dbSNP
rs570758931	850	dbSNP
rs1369985925	854	dbSNP
rs533242991	857	dbSNP
rs1343127827	863	dbSNP
rs890881354	866	dbSNP
rs1461125712	868	dbSNP
rs549896988	871	dbSNP
rs1162563031	873	dbSNP
rs78309626	877	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.