>TAF12|NM_001135218|3'UTR
   1 CACACGGAAAGGTCAGGGAATGGACAGCAATGTATTTGGAGATACTTGAGCTGAGAACTCAGCCATCTCATCCTTGGATT
  81 TTTTTTTTTAATGCTTTACAGAGAAGCATATATTTTTTATTAACAGTGCAGCAATATCTATAATGACTGAGAGGATCTGC
 161 CAAAAGAATAAAGCCTCCTACCCCAACTTCCGGTCCCTTTTCCCTGCCATCTCAAAGAGGAGCAATGTATCTTCCAGAGA
 241 AGATTTTATTTGTGGTTTATTATATAAGTGACTGAATATGGGACAAAGCATTATGGTCTTTTTGGGTAAGACAGTATTAG
 321 CAGGATTTGTAAAGGGTTTTGTTTCCTTCTCCCTTCCCCTTTTCCCTGTACTTTGTAATGTCAGTGTTTATATGAATATG
 401 ACTTTCATTTGCTTTTCCAGAATAAAGAAGTTATTAATCGAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' caguGGUAGAAGGGAAGa 5' || || ||||||| Target 5' gtttCCTTC-TCCCTTCc 3'	341 - 357	149.00	-13.70
2	miRNA 3' caGUGGUAGA-----AGGGAAGa 5' | ||| || ||||||: Target 5' taCCCCAACTTCCGGTCCCTTTt 3'	179 - 201	132.00	-13.30
3	miRNA 3' caGUGGUAGAAGGGAAGa 5' ||::|| ||::||: Target 5' agCATTATGGTCTTTTTg 3'	287 - 304	108.00	-8.30

Mutant ID	Mutant Position	Mutant Source
COSN511044	54	COSMIC
COSN30164891	55	COSMIC
COSN31567974	85	COSMIC
COSN30493200	86	COSMIC
COSN30146502	103	COSMIC
COSN32052898	110	COSMIC
COSN30129017	112	COSMIC
COSN31573924	112	COSMIC
COSN30102469	138	COSMIC
COSN30193731	153	COSMIC
COSN31569888	166	COSMIC
COSN31545161	178	COSMIC
COSN31576861	366	COSMIC
COSN31551200	382	COSMIC
COSN2473093	420	COSMIC
COSN25461470	564	COSMIC
COSN1102478	594	COSMIC
COSN32065130	609	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs762803090	1	dbSNP
rs773120868	5	dbSNP
rs769663156	6	dbSNP
rs748041995	10	dbSNP
rs780131084	19	dbSNP
rs1417256520	23	dbSNP
rs181683597	24	dbSNP
rs1381951368	27	dbSNP
rs1338906915	39	dbSNP
rs746070447	43	dbSNP
rs929515571	44	dbSNP
rs367740448	46	dbSNP
rs368234047	47	dbSNP
rs1484983895	54	dbSNP
rs906320624	60	dbSNP
rs917941498	65	dbSNP
rs1290240874	74	dbSNP
rs1311702731	86	dbSNP
rs1025136148	90	dbSNP
rs778671845	90	dbSNP
rs993226068	90	dbSNP
rs1037345479	92	dbSNP
rs938014847	100	dbSNP
rs926735306	107	dbSNP
rs1403847898	119	dbSNP
rs369201316	119	dbSNP
rs1303085348	122	dbSNP
rs941954384	132	dbSNP
rs889016552	141	dbSNP
rs979538033	142	dbSNP
rs1170406929	148	dbSNP
rs1050243255	150	dbSNP
rs1389497372	156	dbSNP
rs1362672830	163	dbSNP
rs1186368114	165	dbSNP
rs933149219	174	dbSNP
rs923075945	179	dbSNP
rs190758495	182	dbSNP
rs754692017	190	dbSNP
rs968960232	191	dbSNP
rs945459819	192	dbSNP
rs913906704	196	dbSNP
rs531198534	197	dbSNP
rs1385640389	200	dbSNP
rs1297549103	205	dbSNP
rs185580700	208	dbSNP
rs1342682439	209	dbSNP
rs1312947965	219	dbSNP
rs1182829248	225	dbSNP
rs1373079797	227	dbSNP
rs376739066	229	dbSNP
rs752104583	242	dbSNP
rs1264171487	256	dbSNP
rs1030188505	259	dbSNP
rs1205900524	264	dbSNP
rs958300509	272	dbSNP
rs1188507949	273	dbSNP
rs1485596275	274	dbSNP
rs1258978682	279	dbSNP
rs764663606	281	dbSNP
rs1033928910	297	dbSNP
rs980969855	314	dbSNP
rs1483434580	335	dbSNP
rs763167233	352	dbSNP
rs1274519211	353	dbSNP
rs1194628207	371	dbSNP
rs1358903542	374	dbSNP
rs970893171	380	dbSNP
rs546183580	383	dbSNP
rs993670172	384	dbSNP
rs961790936	389	dbSNP
rs1409852128	392	dbSNP
rs1349392896	396	dbSNP
rs532799460	402	dbSNP
rs964405530	404	dbSNP
rs1326738082	405	dbSNP
rs1016370084	406	dbSNP
rs1017771996	408	dbSNP
rs1328536250	420	dbSNP
rs1297320066	427	dbSNP
rs1382392258	430	dbSNP
rs137924337	435	dbSNP
rs887958143	440	dbSNP
rs1286543830	444	dbSNP
rs4252996	449	dbSNP
rs181321114	452	dbSNP
rs1251260518	453	dbSNP
rs566316949	458	dbSNP
rs1047902504	463	dbSNP
rs753691922	473	dbSNP
rs886961080	476	dbSNP
rs143515983	480	dbSNP
rs993646572	481	dbSNP
rs1333178208	484	dbSNP
rs901693957	488	dbSNP
rs1422545830	490	dbSNP
rs1041524381	495	dbSNP
rs896769563	496	dbSNP
rs188117263	497	dbSNP
rs1194182164	508	dbSNP
rs1428071728	523	dbSNP
rs913969955	524	dbSNP
rs573780093	526	dbSNP
rs1054235722	550	dbSNP
rs1259904445	555	dbSNP
rs936647372	558	dbSNP
rs926576166	563	dbSNP
rs1458017727	577	dbSNP
rs1407543995	581	dbSNP
rs1156821597	588	dbSNP
rs981022118	597	dbSNP
rs1378671005	602	dbSNP
rs938051186	604	dbSNP
rs1467392859	615	dbSNP
rs1250368668	622	dbSNP
rs905153374	623	dbSNP
rs1291736698	626	dbSNP
rs1220857551	627	dbSNP
rs761037651	644	dbSNP
rs546877219	645	dbSNP
rs1281707026	653	dbSNP
rs972243139	657	dbSNP
rs769870426	658	dbSNP
rs1016011087	659	dbSNP
rs1005894031	662	dbSNP
rs1320911553	678	dbSNP
rs953054629	687	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.