ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ggggggaggGGCCCUGGg 5' |||||||| Target 5' gtggtagagCCGGGACCc 3'	737 - 754	145.00	-21.30
2	miRNA 3' ggggggagggGCCCUGGg 5' ||||:|| Target 5' gagtgggaggCGGGGCCa 3'	348 - 365	124.00	-14.50
3	miRNA 3' ggggGGA---GGGGCCCUGGg 5' ||| |||| ||:|| Target 5' tgggCCTGGGCCCCAGGGCCa 3'	552 - 572	110.00	-20.90

Mutant ID	Mutant Position	Mutant Source
COSM7696915	43	COSMIC
COSM9042241	57	COSMIC
COSM4879745	60	COSMIC
COSM4388211	78	COSMIC
COSM5964850	81	COSMIC
COSM7683009	100	COSMIC
COSM5795555	121	COSMIC
COSM8852776	139	COSMIC
COSN30118891	166	COSMIC
COSN21444999	175	COSMIC
COSN17211194	226	COSMIC
COSN25883577	446	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs370779067	2	dbSNP
rs928631275	4	dbSNP
rs747922084	5	dbSNP
rs778565211	9	dbSNP
rs924262810	16	dbSNP
rs1186086559	20	dbSNP
rs755003887	21	dbSNP
rs368983834	23	dbSNP
rs767723880	24	dbSNP
rs978426731	25	dbSNP
rs757371498	26	dbSNP
rs751835374	28	dbSNP
rs372203488	35	dbSNP
rs763071257	36	dbSNP
rs753174515	37	dbSNP
rs765488621	38	dbSNP
rs760087374	39	dbSNP
rs777075996	41	dbSNP
rs542692645	48	dbSNP
rs778278574	49	dbSNP
rs749057932	52	dbSNP
rs1459779376	54	dbSNP
rs1350760441	55	dbSNP
rs771818227	56	dbSNP
rs117647993	60	dbSNP
rs778813133	61	dbSNP
rs1189263324	64	dbSNP
rs985906683	66	dbSNP
rs768519192	67	dbSNP
rs749265652	69	dbSNP
rs1257933358	70	dbSNP
rs757424477	71	dbSNP
rs751717997	72	dbSNP
rs1250967553	75	dbSNP
rs777709785	78	dbSNP
rs1339612067	79	dbSNP
rs1276279866	81	dbSNP
rs957550178	88	dbSNP
rs756257806	92	dbSNP
rs1428563507	95	dbSNP
rs553941215	99	dbSNP
rs1389027885	100	dbSNP
rs750725427	102	dbSNP
rs1297940672	108	dbSNP
rs905198957	109	dbSNP
rs765658414	110	dbSNP
rs1372675232	118	dbSNP
rs1045524341	123	dbSNP
rs34124904	125	dbSNP
rs781235865	130	dbSNP
rs540571414	135	dbSNP
rs754349659	136	dbSNP
rs1375935667	137	dbSNP
rs753553661	138	dbSNP
rs1434826154	155	dbSNP
rs1255862298	160	dbSNP
rs578164529	161	dbSNP
rs1267861263	167	dbSNP
rs1181156584	168	dbSNP
rs1039593	171	dbSNP
rs187031975	172	dbSNP
rs761160778	173	dbSNP
rs1252156676	174	dbSNP
rs569527921	174	dbSNP
rs1253726043	175	dbSNP
rs766217878	176	dbSNP
rs771455971	178	dbSNP
rs1220014440	180	dbSNP
rs761547262	181	dbSNP
rs1313265635	183	dbSNP
rs1192360019	184	dbSNP
rs183221431	189	dbSNP
rs112593209	190	dbSNP
rs1170675957	191	dbSNP
rs1352655944	197	dbSNP
rs1308871572	201	dbSNP
rs567515154	203	dbSNP
rs1290419170	208	dbSNP
rs1162079549	209	dbSNP
rs1365430165	210	dbSNP
rs1005322331	218	dbSNP
rs1295862906	226	dbSNP
rs553637970	240	dbSNP
rs34186490	243	dbSNP
rs986260570	243	dbSNP
rs1367646867	246	dbSNP
rs1318197074	260	dbSNP
rs527600383	267	dbSNP
rs148959185	279	dbSNP
rs551508946	280	dbSNP
rs1345938331	290	dbSNP
rs1223652087	293	dbSNP
rs1258938991	305	dbSNP
rs74834302	311	dbSNP
rs1403030425	312	dbSNP
rs562614637	323	dbSNP
rs906452525	325	dbSNP
rs536523852	337	dbSNP
rs946070470	340	dbSNP
rs913302635	342	dbSNP
rs1167490768	343	dbSNP
rs1343473056	344	dbSNP
rs1047029	359	dbSNP
rs879308756	361	dbSNP
rs1470396007	365	dbSNP
rs1391020580	379	dbSNP
rs3741366	380	dbSNP
rs1314696738	381	dbSNP
rs1379592736	391	dbSNP
rs1229857524	394	dbSNP
rs1174662891	397	dbSNP
rs905167514	406	dbSNP
rs529299355	408	dbSNP
rs560320262	410	dbSNP
rs1316287254	411	dbSNP
rs911369160	420	dbSNP
rs1215802803	421	dbSNP
rs367972776	422	dbSNP
rs201724518	425	dbSNP
rs1263134007	426	dbSNP
rs11227466	427	dbSNP
rs116864252	430	dbSNP
rs1014069119	440	dbSNP
rs751350402	447	dbSNP
rs1486590262	449	dbSNP
rs922877254	449	dbSNP
rs896598337	454	dbSNP
rs761438578	458	dbSNP
rs1419008233	468	dbSNP
rs889683314	474	dbSNP
rs1414255374	483	dbSNP
rs1468340011	484	dbSNP
rs1379603838	495	dbSNP
rs961051346	498	dbSNP
rs903032186	499	dbSNP
rs1359993134	504	dbSNP
rs1449890145	520	dbSNP
rs1264783327	522	dbSNP
rs1221520704	529	dbSNP
rs764137872	533	dbSNP
rs1372437627	534	dbSNP
rs1228919408	538	dbSNP
rs537860035	539	dbSNP
rs192877986	540	dbSNP
rs947324633	541	dbSNP
rs1216230642	543	dbSNP
rs879298207	548	dbSNP
rs3177514	549	dbSNP
rs556194094	550	dbSNP
rs116183086	555	dbSNP
rs933055067	556	dbSNP
rs567330411	559	dbSNP
rs1003823041	564	dbSNP
rs1254639837	570	dbSNP
rs753102720	571	dbSNP
rs971808403	584	dbSNP
rs1334967433	591	dbSNP
rs962172163	596	dbSNP
rs1469650364	606	dbSNP
rs145780180	610	dbSNP
rs1353687253	616	dbSNP
rs985019875	626	dbSNP
rs1309732698	627	dbSNP
rs1343589459	632	dbSNP
rs1274930728	637	dbSNP
rs969692434	637	dbSNP
rs1024044695	639	dbSNP
rs1427219480	640	dbSNP
rs1010229948	643	dbSNP
rs1187320981	649	dbSNP
rs891773471	668	dbSNP
rs1461367215	669	dbSNP
rs1206349758	674	dbSNP
rs149792941	686	dbSNP
rs936031146	687	dbSNP
rs570066211	690	dbSNP
rs1199416709	693	dbSNP
rs1482830751	695	dbSNP
rs1247353766	696	dbSNP
rs1479076958	713	dbSNP
rs1251620573	727	dbSNP
rs961074068	728	dbSNP
rs1229555891	731	dbSNP
rs1049915233	733	dbSNP
rs1430113725	736	dbSNP
rs188481560	737	dbSNP
rs749286699	745	dbSNP
rs1423922762	746	dbSNP
rs922858941	747	dbSNP
rs1428085505	748	dbSNP
rs756859035	748	dbSNP
rs972987223	756	dbSNP
rs551853990	757	dbSNP
rs902999760	759	dbSNP
rs1042894633	760	dbSNP
rs909514737	765	dbSNP
rs1209744713	768	dbSNP
rs1452127580	770	dbSNP
rs983761635	776	dbSNP
rs532108020	794	dbSNP
rs1300195814	800	dbSNP
rs1241685250	801	dbSNP
rs959147456	814	dbSNP
rs1460931969	827	dbSNP
rs183547244	830	dbSNP
rs1190824042	839	dbSNP
rs1383139311	842	dbSNP
rs932960585	844	dbSNP
rs1164766736	845	dbSNP
rs922939269	853	dbSNP
rs1003359845	856	dbSNP
rs746022505	857	dbSNP
rs537742544	878	dbSNP
rs940442138	885	dbSNP
rs1386531484	891	dbSNP
rs759717168	896	dbSNP
rs1381000966	902	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.