ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ucUGGGGAGGAGGUAGg 5' |||||| ||||||| Target 5' ggACCCCT-CTCCATCt 3'	325 - 340	162.00	-25.50
2	miRNA 3' ucuGGGGAG-GAGGUAGg 5' || :|| :|||||| Target 5' tttCCATTCATTCCATCt 3'	373 - 390	134.00	-13.40
3	miRNA 3' ucUGGGGA-GGAGGUAGg 5' :::||| |||||| | Target 5' atGTTCCTCCCTCCAACt 3'	35 - 52	123.00	-15.70

Mutant ID	Mutant Position	Mutant Source
COSM1210026	4	COSMIC
COSM8425401	9	COSMIC
COSM4614857	12	COSMIC
COSM6588683	20	COSMIC
COSM1395617	32	COSMIC
COSM4559164	97	COSMIC
COSM7708205	113	COSMIC
COSM4619494	162	COSMIC
COSM4615642	177	COSMIC
COSM393112	184	COSMIC
COSM4617996	195	COSMIC
COSM7591781	204	COSMIC
COSM7416927	211	COSMIC
COSM7295723	232	COSMIC
COSM4632816	242	COSMIC
COSN30592887	255	COSMIC
COSN20079509	311	COSMIC
COSN30176942	315	COSMIC
COSN30549241	346	COSMIC
COSN30632900	406	COSMIC
COSN14966313	468	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1354564384	2	dbSNP
rs762177710	4	dbSNP
rs1020004431	6	dbSNP
rs1323343683	6	dbSNP
rs1467553605	7	dbSNP
rs765805593	8	dbSNP
rs750688958	9	dbSNP
rs552868043	10	dbSNP
rs967546045	12	dbSNP
rs1222645552	14	dbSNP
rs1281550956	15	dbSNP
rs1349550824	16	dbSNP
rs1211117204	18	dbSNP
rs757885964	19	dbSNP
rs1000335820	20	dbSNP
rs940549564	21	dbSNP
rs201052217	22	dbSNP
rs1235048455	23	dbSNP
rs866790411	30	dbSNP
rs1470813497	31	dbSNP
rs544743697	32	dbSNP
rs368644338	33	dbSNP
rs781227352	36	dbSNP
rs1346755906	43	dbSNP
rs748278231	44	dbSNP
rs1156555700	45	dbSNP
rs1395883532	46	dbSNP
rs371623584	51	dbSNP
rs1412621896	52	dbSNP
rs1330649189	57	dbSNP
rs1024343750	58	dbSNP
rs1431496194	60	dbSNP
rs971803111	64	dbSNP
rs1045080688	66	dbSNP
rs1441427752	69	dbSNP
rs1205011010	70	dbSNP
rs1277743730	71	dbSNP
rs1253003069	72	dbSNP
rs1476520491	73	dbSNP
rs1372510344	77	dbSNP
rs373687274	80	dbSNP
rs1001098231	81	dbSNP
rs1279082814	82	dbSNP
rs1345314232	83	dbSNP
rs908601951	87	dbSNP
rs1052624677	94	dbSNP
rs748051957	97	dbSNP
rs1328846627	99	dbSNP
rs973189838	102	dbSNP
rs1480865346	103	dbSNP
rs868141486	105	dbSNP
rs1421823482	106	dbSNP
rs1438799313	113	dbSNP
rs1181699337	116	dbSNP
rs1008395880	117	dbSNP
rs1363936754	118	dbSNP
rs1236949054	119	dbSNP
rs1455636956	121	dbSNP
rs1171626779	127	dbSNP
rs771990238	129	dbSNP
rs367791868	132	dbSNP
rs1456841423	139	dbSNP
rs190801974	140	dbSNP
rs1169389497	141	dbSNP
rs1377152533	151	dbSNP
rs890129579	152	dbSNP
rs769857104	156	dbSNP
rs752423341	159	dbSNP
rs1326764918	161	dbSNP
rs1373103477	162	dbSNP
rs1486197105	163	dbSNP
rs768445779	165	dbSNP
rs1406293544	167	dbSNP
rs1302093037	168	dbSNP
rs1212588695	170	dbSNP
rs530306798	171	dbSNP
rs1368289051	173	dbSNP
rs1254929850	176	dbSNP
rs183044011	179	dbSNP
rs902910661	181	dbSNP
rs761394201	182	dbSNP
rs914366504	184	dbSNP
rs765607052	185	dbSNP
rs1470229815	186	dbSNP
rs1214341881	192	dbSNP
rs566920318	195	dbSNP
rs1431549573	196	dbSNP
rs763515779	198	dbSNP
rs1373912440	204	dbSNP
rs1033208052	211	dbSNP
rs1314421194	212	dbSNP
rs766695241	213	dbSNP
rs1168290381	215	dbSNP
rs1401717446	216	dbSNP
rs867764533	220	dbSNP
rs1220789666	222	dbSNP
rs1291073948	223	dbSNP
rs3760768	233	dbSNP
rs751966512	236	dbSNP
rs1260073165	240	dbSNP
rs1325187759	243	dbSNP
rs1490528674	246	dbSNP
rs1391628830	247	dbSNP
rs1383476493	248	dbSNP
rs1318566730	249	dbSNP
rs1325488801	250	dbSNP
rs1227072533	251	dbSNP
rs1276719849	252	dbSNP
rs1440372284	254	dbSNP
rs1337531010	255	dbSNP
rs1195930518	256	dbSNP
rs1234401643	256	dbSNP
rs1273161247	258	dbSNP
rs532663451	259	dbSNP
rs1255526482	263	dbSNP
rs1206979115	266	dbSNP
rs1485671193	267	dbSNP
rs1262623880	271	dbSNP
rs1187400571	274	dbSNP
rs1294938203	276	dbSNP
rs1446660848	281	dbSNP
rs1191190014	282	dbSNP
rs1024939759	287	dbSNP
rs1340149879	292	dbSNP
rs1475443019	294	dbSNP
rs1329777256	297	dbSNP
rs1164118498	300	dbSNP
rs1366720005	301	dbSNP
rs1415183523	305	dbSNP
rs1330697608	306	dbSNP
rs1462453202	310	dbSNP
rs948204653	317	dbSNP
rs552658327	322	dbSNP
rs982719401	329	dbSNP
rs1157099803	334	dbSNP
rs187571419	345	dbSNP
rs113160292	346	dbSNP
rs1472665078	348	dbSNP
rs769777076	350	dbSNP
rs1015652164	351	dbSNP
rs1235853302	360	dbSNP
rs866468973	382	dbSNP
rs936689565	386	dbSNP
rs962794469	387	dbSNP
rs1052344911	392	dbSNP
rs1355374756	399	dbSNP
rs892725086	399	dbSNP
rs569286407	400	dbSNP
rs1449983027	401	dbSNP
rs1041252737	404	dbSNP
rs920476262	405	dbSNP
rs899981180	409	dbSNP
rs75765597	418	dbSNP
rs1386150227	422	dbSNP
rs554997634	423	dbSNP
rs986071969	427	dbSNP
rs1160607951	436	dbSNP
rs911768900	453	dbSNP
rs1378064325	459	dbSNP
rs113690946	462	dbSNP
rs567576698	464	dbSNP
rs1454323270	467	dbSNP
rs1005893551	469	dbSNP
rs534011465	470	dbSNP
rs1014970783	476	dbSNP
rs962021601	477	dbSNP
rs1232838625	502	dbSNP
rs1285560198	509	dbSNP
rs11554339	510	dbSNP
rs1277785815	512	dbSNP
rs1443736311	519	dbSNP
rs902838306	531	dbSNP
rs1212740372	538	dbSNP
rs770771592	539	dbSNP
rs1436418906	552	dbSNP
rs1327491078	553	dbSNP
rs1320737908	562	dbSNP
rs1358299173	563	dbSNP
rs1418228846	563	dbSNP
rs374998691	573	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.