ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cuuGUAUUGUGCCU-AUUGGAga 5' || || | | |||||| Target 5' tgcCAGAAAAGTCACTAACCTgt 3'	28 - 50	127.00	-7.40
2	miRNA 3' cuUGUA--UUGUGCCUAUUGGAga 5' |||| :| || |:|::||| Target 5' agACATGCGAGACTGGTGGCCTgc 3'	183 - 206	114.00	-12.40
3	miRNA 3' cuuguAUU-GUGC---CUAUUG--GAGa 5' ||| :::| |||||| ||| Target 5' agggtTAAGTGTGCAAGATAACTACTCg 3'	525 - 552	113.00	-8.90

Mutant ID	Mutant Position	Mutant Source
348153	13	ClinVar
348152	31	ClinVar
899738	46	ClinVar
903355	103	ClinVar
903354	187	ClinVar
348151	220	ClinVar
348149	221	ClinVar
348150	221	ClinVar
348148	247	ClinVar
903353	277	ClinVar
348147	278	ClinVar
348146	329	ClinVar
902489	374	ClinVar
348145	403	ClinVar
348144	408	ClinVar
348143	450	ClinVar
348142	468	ClinVar
348141	510	ClinVar
348140	568	ClinVar
900816	612	ClinVar
348139	622	ClinVar
348138	682	ClinVar
348137	690	ClinVar
COSN30515906	5	COSMIC
COSN30154085	10	COSMIC
COSN30124265	16	COSMIC
COSN26574348	89	COSMIC
COSN30535683	96	COSMIC
COSN21296456	114	COSMIC
COSN30137604	119	COSMIC
COSN5561946	146	COSMIC
COSN20075042	168	COSMIC
COSN26435537	190	COSMIC
COSN31586112	191	COSMIC
COSN25910991	205	COSMIC
COSN30111299	230	COSMIC
COSN20099832	244	COSMIC
COSN17859727	638	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs748449867	1	dbSNP
rs1470596714	4	dbSNP
rs868457361	6	dbSNP
rs779024950	7	dbSNP
rs1426865196	12	dbSNP
rs536397999	13	dbSNP
rs768398633	15	dbSNP
rs749095312	16	dbSNP
rs551937467	18	dbSNP
rs1375204306	22	dbSNP
rs1462813957	25	dbSNP
rs1197598431	27	dbSNP
rs1436130389	29	dbSNP
rs377243912	31	dbSNP
rs780805628	34	dbSNP
rs567249048	46	dbSNP
rs138092282	47	dbSNP
rs756455772	47	dbSNP
rs371229754	55	dbSNP
rs1010432180	58	dbSNP
rs529161920	68	dbSNP
rs1257395251	75	dbSNP
rs1441104345	77	dbSNP
rs893162896	82	dbSNP
rs1253644050	83	dbSNP
rs1488192107	88	dbSNP
rs1333469240	92	dbSNP
rs1304469735	93	dbSNP
rs117036858	103	dbSNP
rs1001640559	104	dbSNP
rs936732192	107	dbSNP
rs1422148800	114	dbSNP
rs1463414939	119	dbSNP
rs1163543868	125	dbSNP
rs1403887426	133	dbSNP
rs1428192081	139	dbSNP
rs1435121315	140	dbSNP
rs753015043	141	dbSNP
rs1363603600	147	dbSNP
rs903896010	159	dbSNP
rs905909541	168	dbSNP
rs1277285769	174	dbSNP
rs1388036988	188	dbSNP
rs759748053	190	dbSNP
rs1316007101	203	dbSNP
rs1410636786	208	dbSNP
rs1235126795	212	dbSNP
rs543249045	217	dbSNP
rs547449704	218	dbSNP
rs1049581	220	dbSNP
rs1049582	221	dbSNP
rs386672163	222	dbSNP
rs540669318	222	dbSNP
rs879630614	222	dbSNP
rs1193403202	227	dbSNP
rs1036307949	232	dbSNP
rs1201624985	246	dbSNP
rs71810044	247	dbSNP
rs1268016430	256	dbSNP
rs1189352699	265	dbSNP
rs908894896	266	dbSNP
rs1404290849	267	dbSNP
rs1162030200	269	dbSNP
rs761236174	270	dbSNP
rs765235173	278	dbSNP
rs1284772128	285	dbSNP
rs1338238888	289	dbSNP
rs1416363498	302	dbSNP
rs1284764537	306	dbSNP
rs938186006	327	dbSNP
rs886059238	329	dbSNP
rs1309152144	330	dbSNP
rs1354709857	334	dbSNP
rs1216235131	352	dbSNP
rs979852903	352	dbSNP
rs975798448	355	dbSNP
rs1212016141	356	dbSNP
rs1237430197	358	dbSNP
rs372587697	360	dbSNP
rs952459308	361	dbSNP
rs966211522	364	dbSNP
rs1348157372	392	dbSNP
rs11556811	402	dbSNP
rs1049600	403	dbSNP
rs1049601	408	dbSNP
rs1157722660	417	dbSNP
rs961041392	418	dbSNP
rs1436381359	420	dbSNP
rs1435020092	425	dbSNP
rs1033021934	442	dbSNP
rs886059237	450	dbSNP
rs1400514067	456	dbSNP
rs1001901740	466	dbSNP
rs699460	468	dbSNP
rs1008106052	470	dbSNP
rs889632121	471	dbSNP
rs1377825309	485	dbSNP
rs1239473297	490	dbSNP
rs1203323520	496	dbSNP
rs1307404791	498	dbSNP
rs575755381	506	dbSNP
rs1031327	510	dbSNP
rs1000975076	511	dbSNP
rs1014332876	516	dbSNP
rs769332015	518	dbSNP
rs536485721	527	dbSNP
rs1177416578	531	dbSNP
rs940599171	533	dbSNP
rs1455255754	534	dbSNP
rs945389981	542	dbSNP
rs567445899	550	dbSNP
rs181809154	551	dbSNP
rs1242461778	552	dbSNP
rs386672162	556	dbSNP
rs1412538022	566	dbSNP
rs1330855714	567	dbSNP
rs560933456	568	dbSNP
rs1445093980	573	dbSNP
rs926830522	576	dbSNP
rs931675986	584	dbSNP
rs984904012	585	dbSNP
rs41268391	596	dbSNP
rs919682064	600	dbSNP
rs975828016	600	dbSNP
rs571631133	606	dbSNP
rs913415768	611	dbSNP
rs776067259	612	dbSNP
rs988905734	618	dbSNP
rs1452825054	619	dbSNP
rs768383773	621	dbSNP
rs10604	622	dbSNP
rs762791839	626	dbSNP
rs1290001887	629	dbSNP
rs1424286306	638	dbSNP
rs746985958	645	dbSNP
rs531790880	647	dbSNP
rs1396852295	648	dbSNP
rs980371371	655	dbSNP
rs986754392	656	dbSNP
rs1329513602	663	dbSNP
rs1433368137	681	dbSNP
rs699459	682	dbSNP
rs1340467727	687	dbSNP
rs189439260	689	dbSNP
rs1031326	690	dbSNP
rs1014363903	695	dbSNP
rs1000715019	707	dbSNP
rs1209886546	730	dbSNP
rs1288726022	737	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.