ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cgGGAGG---GGACUGAGGGACu 5' ||||| ||| ||||||| Target 5' ctCCTCCATTCCTCTCTCCCTGc 3'	323 - 345	162.00	-25.40
2	miRNA 3' cgGGAGGGGACUGAGGGACu 5' || ||||| ||||||| Target 5' gcCCCCCCCT-TCTCCCTGa 3'	212 - 230	161.00	-31.50
3	miRNA 3' cgGGAGG-GGA--CUGAGGGAcu 5' ||||| ||| | |||||| Target 5' ctCCTCCACCTGCGCCTCCCTtg 3'	109 - 131	144.00	-25.90

Mutant ID	Mutant Position	Mutant Source
COSN30528116	2	COSMIC
COSN7450328	99	COSMIC
COSN20079566	116	COSMIC
COSN22451842	206	COSMIC
COSN27493771	392	COSMIC
COSN29268706	510	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1252698746	3	dbSNP
rs1477964486	4	dbSNP
rs1179483175	5	dbSNP
rs1196264789	6	dbSNP
rs754707088	8	dbSNP
rs779115309	10	dbSNP
rs996350859	11	dbSNP
rs1197435811	12	dbSNP
rs748588681	14	dbSNP
rs1412822969	15	dbSNP
rs1029603173	17	dbSNP
rs538483363	18	dbSNP
rs370843874	24	dbSNP
rs1456848969	25	dbSNP
rs756663802	27	dbSNP
rs1324472545	28	dbSNP
rs552871613	30	dbSNP
rs747020703	31	dbSNP
rs770948744	32	dbSNP
rs1423534201	33	dbSNP
rs1208055861	35	dbSNP
rs1311833251	39	dbSNP
rs1279049064	40	dbSNP
rs1419777570	40	dbSNP
rs1386487802	41	dbSNP
rs1156801472	44	dbSNP
rs776867150	45	dbSNP
rs981895600	46	dbSNP
rs537440349	49	dbSNP
rs1428310127	50	dbSNP
rs937457690	55	dbSNP
rs991326139	59	dbSNP
rs750000280	62	dbSNP
rs1424581088	63	dbSNP
rs931197230	68	dbSNP
rs1203939437	69	dbSNP
rs1350630914	70	dbSNP
rs554560510	76	dbSNP
rs758088529	77	dbSNP
rs886732523	78	dbSNP
rs1333058717	86	dbSNP
rs60367783	87	dbSNP
rs777166885	87	dbSNP
rs1041141167	88	dbSNP
rs899566911	93	dbSNP
rs1315573178	96	dbSNP
rs1458489367	103	dbSNP
rs540945373	106	dbSNP
rs554039855	107	dbSNP
rs1166927928	110	dbSNP
rs978733634	112	dbSNP
rs1416884794	115	dbSNP
rs1164546599	118	dbSNP
rs922022272	119	dbSNP
rs778198743	120	dbSNP
rs10407167	123	dbSNP
rs890660779	128	dbSNP
rs1011750371	130	dbSNP
rs147321718	131	dbSNP
rs967644127	141	dbSNP
rs981460136	143	dbSNP
rs1036130857	146	dbSNP
rs1341075590	147	dbSNP
rs562967507	149	dbSNP
rs1221749459	151	dbSNP
rs186782645	157	dbSNP
rs1327912728	159	dbSNP
rs1438601330	160	dbSNP
rs1393515080	171	dbSNP
rs992003459	180	dbSNP
rs1026580232	182	dbSNP
rs1484881636	192	dbSNP
rs1206644460	193	dbSNP
rs919722115	194	dbSNP
rs905627986	196	dbSNP
rs1263582624	202	dbSNP
rs1001278441	209	dbSNP
rs558937136	213	dbSNP
rs931135403	213	dbSNP
rs960172989	213	dbSNP
rs189978566	215	dbSNP
rs908450030	217	dbSNP
rs182564045	218	dbSNP
rs79646647	219	dbSNP
rs373873984	220	dbSNP
rs1376185028	221	dbSNP
rs1429324663	221	dbSNP
rs920947180	222	dbSNP
rs1424456774	227	dbSNP
rs544730030	235	dbSNP
rs1178279647	238	dbSNP
rs1231510469	240	dbSNP
rs145803616	240	dbSNP
rs1471394676	240	dbSNP
rs575992555	240	dbSNP
rs1343913049	241	dbSNP
rs775918921	242	dbSNP
rs1045287106	244	dbSNP
rs1284951317	246	dbSNP
rs1278703158	247	dbSNP
rs905570941	247	dbSNP
rs377592024	249	dbSNP
rs1356361645	252	dbSNP
rs1314935790	255	dbSNP
rs1433013720	261	dbSNP
rs1358639999	262	dbSNP
rs274154	267	dbSNP
rs1402923219	268	dbSNP
rs1413326634	275	dbSNP
rs1011699415	279	dbSNP
rs895540870	282	dbSNP
rs1010496431	284	dbSNP
rs1185337325	284	dbSNP
rs1020079950	285	dbSNP
rs1217914158	287	dbSNP
rs968623331	287	dbSNP
rs1000155870	288	dbSNP
rs1233687325	289	dbSNP
rs1291052109	289	dbSNP
rs568571971	290	dbSNP
rs138024889	294	dbSNP
rs1305723387	295	dbSNP
rs1193983270	300	dbSNP
rs1314649882	301	dbSNP
rs953389298	302	dbSNP
rs1432921355	308	dbSNP
rs1286672165	312	dbSNP
rs1343965375	313	dbSNP
rs903434774	323	dbSNP
rs1158993321	347	dbSNP
rs1003561354	348	dbSNP
rs1035674050	354	dbSNP
rs987473023	361	dbSNP
rs958973485	382	dbSNP
rs1162406096	394	dbSNP
rs1208552576	394	dbSNP
rs1012961762	395	dbSNP
rs537552319	400	dbSNP
rs962125091	403	dbSNP
rs1187787028	410	dbSNP
rs8419	412	dbSNP
rs952805610	413	dbSNP
rs982597963	416	dbSNP
rs908397722	417	dbSNP
rs1232412519	421	dbSNP
rs1176867637	423	dbSNP
rs1321377799	427	dbSNP
rs761837788	428	dbSNP
rs1420968232	429	dbSNP
rs775197386	431	dbSNP
rs1045318630	432	dbSNP
rs1320538447	438	dbSNP
rs1046144	442	dbSNP
rs1397188227	444	dbSNP
rs937020402	446	dbSNP
rs1373301878	461	dbSNP
rs1056770067	466	dbSNP
rs533585375	473	dbSNP
rs932311839	474	dbSNP
rs1388991586	476	dbSNP
rs1053425074	479	dbSNP
rs540896098	483	dbSNP
rs914985094	484	dbSNP
rs1209039433	486	dbSNP
rs1480800811	491	dbSNP
rs904394173	494	dbSNP
rs1206869424	500	dbSNP
rs1448252830	502	dbSNP
rs1305883072	503	dbSNP
rs945158726	506	dbSNP
rs1000018935	508	dbSNP
rs760544971	510	dbSNP
rs903382530	515	dbSNP
rs1386568189	520	dbSNP
rs554423571	521	dbSNP
rs11268713	527	dbSNP
rs149503358	527	dbSNP
rs577658438	539	dbSNP
rs894763993	540	dbSNP
rs1309885808	541	dbSNP
rs1455152080	546	dbSNP
rs1393205435	548	dbSNP
rs1198078902	555	dbSNP
rs1419510510	557	dbSNP
rs1252373174	559	dbSNP
rs560753959	560	dbSNP
rs962442423	560	dbSNP
rs546409316	563	dbSNP
rs1046169	565	dbSNP
rs1211700245	577	dbSNP
rs1350059349	579	dbSNP
rs1282142696	580	dbSNP
rs1278895525	581	dbSNP
rs1013323428	588	dbSNP
rs1027682135	589	dbSNP
rs1309648762	591	dbSNP
rs1389982924	595	dbSNP
rs1027014250	603	dbSNP
rs1046170	609	dbSNP
rs1046174	611	dbSNP
rs749923882	615	dbSNP
rs1231399481	618	dbSNP
rs1481655854	620	dbSNP
rs1004317474	622	dbSNP
rs1373084614	623	dbSNP
rs74756334	624	dbSNP
rs965594798	634	dbSNP
rs1156691567	639	dbSNP
rs1453162583	643	dbSNP
rs1472108370	647	dbSNP
rs1176113501	653	dbSNP
rs1188918188	653	dbSNP
rs1426254634	657	dbSNP
rs1409767703	659	dbSNP
rs1169232926	661	dbSNP
rs1046180	663	dbSNP
rs1352473182	667	dbSNP
rs1448341709	670	dbSNP
rs1331483706	673	dbSNP
rs939583222	675	dbSNP
rs1262955717	677	dbSNP
rs576555651	679	dbSNP
rs1293594618	688	dbSNP
rs976657802	690	dbSNP
rs766005532	691	dbSNP
rs916959917	693	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.