>HNRNPF|NM_001098204|3'UTR
   1 TTTTGTTAGGAACATTTGAGTTACTTCAATCATTTTCACAGGCAGCCAACAAGCAATTAAGAGCAGTTATAATAGAGGAA
  81 GCTGGGGGACCCATTTTGCACCATGAGTTTGTGAAAAATCTGGATTAAAAAATTACCTCTTCAGTGTTTTCTCATGCAAA
 161 ATTTTCTTCTAGCATGTGATAATGAGTAAACTAAAACTATTTTCAGCTTTTCTCAATTAACATTTTGGTAGTATACTTCA
 241 GAGTGATGTTATCTAAGTTTAAGTAGTTTAAGTATGTTAAATGTGGATCTTTTACACCACATCACAGTGAACACACTGGG
 321 GAGACGTGCTTTTTTGGAAAACTCAAAGGTGCTAGCTCCCTGATTCAAAGAAATATTTCTCATGTTTGTTCATTCTAGTT
 401 TATATTTTCATTTAAAATCCTTTAGGTTAAGTTTAAGCTTTTTAAAAGTTAGTTTTGAGAATTGAGACACAATACTAATA
 481 CTGTAGGAATTGGTGAGGCCTTGACTTAAAACTTTCTTTGTACTGTGATTTCCTTTTGGGTGTATTTTGCTAAGTGAAAC
 561 TTGTTAAATTTTTTGTTAACTAAATTTTTTTCTTAAAATAAAGACTTTTTCACAATGACTGGCACAGATTACTCAGCAAA
 641 AGATAGCAAAACGGGTGGTTGAAGATAATTCATTTTAATCGTAATGTATTTTAGTGTGAATTTAAAAATTTCATACATCA
 721 AATCTATGATCTCCCTTATATTCTTATGATGAGGCTAAATAAAAGTCTAATAAAAATGTTAAATATGTGAATGGTGGAAA
 801 TGGTGACTAGCAGCACACATTCTGGGAAGCATCAAATAGACACACGGCCCCAGCCACCTGCAACTTATGTGCACTGTTGT
 881 AAACCATTCAGAATTTTCCTGCTAGGCCCTTGATGCTGGAGTCACATCTGTTGATAGCTGGAGAACTTTAGTTTCAAGTA
 961 CTACATTGTGAAAGCAATTATTTTGTTTCTAGTTTTTATAAAATGCTGATTTAAACTTTTAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' acaaUUACGAUUAU-ACAUCCUc 5' ||| |||||| ||||||| Target 5' acacAATACTAATACTGTAGGAa 3'	467 - 489	174.00	-13.30
2	miRNA 3' acAAU---UACGAUUAUACAUCCUc 5' ||| |||:||| |||| ||| Target 5' gtTTAAGTATGTTAA-ATGT-GGAt 3'	266 - 288	119.00	-8.60
3	miRNA 3' acaaUUACGA-UUAUACAUccuc 5' ||||:| |||||||: Target 5' taaaAATGTTAAATATGTGaatg 3'	771 - 793	102.00	-9.00

Mutant ID	Mutant Position	Mutant Source
COSN30104195	23	COSMIC
COSN30170288	28	COSMIC
COSN1119064	29	COSMIC
COSN31601473	51	COSMIC
COSN30505416	53	COSMIC
COSN30493007	71	COSMIC
COSN30156489	75	COSMIC
COSN30502598	106	COSMIC
COSN31486379	115	COSMIC
COSN30501466	145	COSMIC
COSN26559180	172	COSMIC
COSN8640081	184	COSMIC
COSN31488328	204	COSMIC
COSN6970810	511	COSMIC
COSN30174358	556	COSMIC
COSN31568290	567	COSMIC
COSN23299632	594	COSMIC
COSN15328482	714	COSMIC
COSN28665661	730	COSMIC
COSN10067896	1008	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs752284116	1	dbSNP
rs374231673	4	dbSNP
rs1367721885	8	dbSNP
rs201748393	14	dbSNP
rs1425251705	16	dbSNP
rs1269297363	19	dbSNP
rs1336862203	20	dbSNP
rs1247978662	22	dbSNP
rs745877705	24	dbSNP
rs1274731018	28	dbSNP
rs199696858	29	dbSNP
rs768045050	33	dbSNP
rs1313644598	37	dbSNP
rs576923654	39	dbSNP
rs765070824	42	dbSNP
rs1192731505	45	dbSNP
rs1356219950	46	dbSNP
rs754143332	48	dbSNP
rs1404031335	49	dbSNP
rs1247422649	50	dbSNP
rs763769612	51	dbSNP
rs1030764966	54	dbSNP
rs1206422821	56	dbSNP
rs1365059240	57	dbSNP
rs371934952	61	dbSNP
rs1423901796	63	dbSNP
rs997579712	65	dbSNP
rs1387190362	70	dbSNP
rs965184555	77	dbSNP
rs1317388781	78	dbSNP
rs1803714	84	dbSNP
rs1265484215	86	dbSNP
rs540504138	89	dbSNP
rs1229189239	94	dbSNP
rs1319813835	99	dbSNP
rs1358160495	103	dbSNP
rs1294663737	105	dbSNP
rs1297466237	114	dbSNP
rs1435473931	118	dbSNP
rs1364181674	120	dbSNP
rs1348193170	124	dbSNP
rs1448907620	136	dbSNP
rs572777763	137	dbSNP
rs1384652830	139	dbSNP
rs1433135732	143	dbSNP
rs1161579794	150	dbSNP
rs759332250	153	dbSNP
rs10409	164	dbSNP
rs1458348125	165	dbSNP
rs1457866355	166	dbSNP
rs1042251940	169	dbSNP
rs1436794815	174	dbSNP
rs1320894897	184	dbSNP
rs1381390051	186	dbSNP
rs1367161876	194	dbSNP
rs1316541208	202	dbSNP
rs1170890391	204	dbSNP
rs535809284	211	dbSNP
rs896130823	212	dbSNP
rs1056547680	214	dbSNP
rs1209590899	221	dbSNP
rs1242822440	222	dbSNP
rs368475745	223	dbSNP
rs1460160333	228	dbSNP
rs770594533	228	dbSNP
rs937764609	234	dbSNP
rs369913142	252	dbSNP
rs1158351347	254	dbSNP
rs1361848301	255	dbSNP
rs144005766	260	dbSNP
rs372890561	265	dbSNP
rs1404818801	266	dbSNP
rs1467936172	268	dbSNP
rs1304031186	274	dbSNP
rs1033078304	275	dbSNP
rs909381087	275	dbSNP
rs1305575784	278	dbSNP
rs1337888317	283	dbSNP
rs1452925441	287	dbSNP
rs1229328831	289	dbSNP
rs1262910274	292	dbSNP
rs983677919	293	dbSNP
rs1267480586	298	dbSNP
rs548983313	301	dbSNP
rs1208660020	306	dbSNP
rs1256637292	308	dbSNP
rs1480389381	311	dbSNP
rs1487473495	317	dbSNP
rs781200413	318	dbSNP
rs766385838	319	dbSNP
rs11548186	325	dbSNP
rs187150871	326	dbSNP
rs923282518	344	dbSNP
rs1428365230	348	dbSNP
rs1229051224	358	dbSNP
rs1479239867	363	dbSNP
rs1341251028	366	dbSNP
rs976512779	376	dbSNP
rs1226486689	381	dbSNP
rs374306171	382	dbSNP
rs551223636	391	dbSNP
rs1411002446	395	dbSNP
rs1282981833	396	dbSNP
rs1386222834	397	dbSNP
rs1344071078	398	dbSNP
rs1388649689	399	dbSNP
rs1302316848	401	dbSNP
rs1307152402	404	dbSNP
rs1202447161	408	dbSNP
rs1253180964	417	dbSNP
rs1483967150	430	dbSNP
rs1017677510	438	dbSNP
rs1267879662	454	dbSNP
rs1310458526	455	dbSNP
rs1430918645	456	dbSNP
rs774638734	462	dbSNP
rs768843772	468	dbSNP
rs979494546	471	dbSNP
rs376095980	472	dbSNP
rs1167650775	475	dbSNP
rs1391500961	478	dbSNP
rs1167057534	479	dbSNP
rs1478809202	482	dbSNP
rs1430925351	483	dbSNP
rs967817362	487	dbSNP
rs771641140	489	dbSNP
rs1438985283	490	dbSNP
rs552631203	492	dbSNP
rs139521821	494	dbSNP
rs1431021734	509	dbSNP
rs1253311069	513	dbSNP
rs1205848913	516	dbSNP
rs1343015583	520	dbSNP
rs529013340	520	dbSNP
rs1009459732	523	dbSNP
rs567144282	533	dbSNP
rs1291018391	545	dbSNP
rs1449804990	572	dbSNP
rs1482662608	575	dbSNP
rs1272242230	578	dbSNP
rs763282242	580	dbSNP
rs1247096575	585	dbSNP
rs775789809	590	dbSNP
rs1184453784	592	dbSNP
rs143891929	592	dbSNP
rs764202527	603	dbSNP
rs1162395015	605	dbSNP
rs117712109	606	dbSNP
rs1165870402	607	dbSNP
rs34445024	611	dbSNP
rs752976985	611	dbSNP
rs147376570	621	dbSNP
rs1322881901	628	dbSNP
rs1366004036	644	dbSNP
rs1451929182	646	dbSNP
rs941105096	649	dbSNP
rs1290266552	652	dbSNP
rs562975768	653	dbSNP
rs551052502	654	dbSNP
rs1316159860	655	dbSNP
rs1206426968	657	dbSNP
rs1411006477	662	dbSNP
rs747600964	667	dbSNP
rs887914279	677	dbSNP
rs1048388721	678	dbSNP
rs1182404440	680	dbSNP
rs929555544	683	dbSNP
rs1474047135	689	dbSNP
rs1370415361	694	dbSNP
rs1306579229	704	dbSNP
rs1407711455	712	dbSNP
rs34807604	712	dbSNP
rs539723067	716	dbSNP
rs532238802	718	dbSNP
rs923557315	722	dbSNP
rs976101434	723	dbSNP
rs181502268	726	dbSNP
rs540022753	729	dbSNP
rs13376803	730	dbSNP
rs1342390471	731	dbSNP
rs1284771687	743	dbSNP
rs1372545996	744	dbSNP
rs1226279675	746	dbSNP
rs142778208	747	dbSNP
rs988404993	753	dbSNP
rs542218456	754	dbSNP
rs1211422949	765	dbSNP
rs1443711198	765	dbSNP
rs1413565248	777	dbSNP
rs190233547	777	dbSNP
rs1232749999	783	dbSNP
rs949052219	790	dbSNP
rs771511594	791	dbSNP
rs747470016	793	dbSNP
rs138369783	794	dbSNP
rs925456870	796	dbSNP
rs749691400	803	dbSNP
rs1189520986	808	dbSNP
rs1353819425	810	dbSNP
rs578192347	811	dbSNP
rs1289959943	822	dbSNP
rs777327422	828	dbSNP
rs867467821	835	dbSNP
rs780230873	836	dbSNP
rs185372679	837	dbSNP
rs1346698987	839	dbSNP
rs976132693	842	dbSNP
rs1016621272	843	dbSNP
rs1271717354	845	dbSNP
rs1270096926	846	dbSNP
rs878862746	859	dbSNP
rs1482330235	860	dbSNP
rs1207976785	863	dbSNP
rs966427836	865	dbSNP
rs1229018499	867	dbSNP
rs114311147	868	dbSNP
rs1287076481	869	dbSNP
rs1478499842	870	dbSNP
rs1450773222	880	dbSNP
rs1417521130	882	dbSNP
rs1427709512	884	dbSNP
rs986234959	885	dbSNP
rs765583727	891	dbSNP
rs1404043990	893	dbSNP
rs80027918	902	dbSNP
rs1026828959	905	dbSNP
rs1233548045	908	dbSNP
rs902017556	909	dbSNP
rs995743104	916	dbSNP
rs1231247938	919	dbSNP
rs752374095	923	dbSNP
rs1015910722	925	dbSNP
rs750602030	926	dbSNP
rs767847250	927	dbSNP
rs1456306287	928	dbSNP
rs1269946933	929	dbSNP
rs1013472603	931	dbSNP
rs1192799330	949	dbSNP
rs1246260614	955	dbSNP
rs1164974316	957	dbSNP
rs1451473042	961	dbSNP
rs528215857	970	dbSNP
rs1390639672	975	dbSNP
rs1457372349	975	dbSNP
rs1161191791	977	dbSNP
rs1392679467	981	dbSNP
rs1398335399	985	dbSNP
rs896174142	989	dbSNP
rs1126709	991	dbSNP
rs1436566276	992	dbSNP
rs548856161	1000	dbSNP
rs1359292629	1001	dbSNP
rs369573297	1006	dbSNP
rs7905676	1010	dbSNP
rs1460161558	1011	dbSNP
rs903604440	1013	dbSNP
rs1189250978	1015	dbSNP
rs1242634050	1016	dbSNP
rs180922191	1020	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.