ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gcugcacccCGCCUCGCAGACCUUc 5' || | | ||||||| Target 5' agagaatttGCACATC-TCTGGAAg 3'	97 - 120	143.00	-11.90
2	miRNA 3' gcUGCACCCCGCCUCGCAGACCUUc 5' |||||| || | | |||| || Target 5' caACGTGGTGC-TATC-TCTGAAAc 3'	216 - 238	133.00	-12.06
3	miRNA 3' gcUGCACCCCG--CCUCGC-----------AGACCUUc 5' ||||||| | | :| |:||||| Target 5' caACGTGGGACATTGTTTGTTTCTTCCATATTTGGAAg 3'	521 - 558	125.00	-21.64

Mutant ID	Mutant Position	Mutant Source
COSN26994521	53	COSMIC
COSN31523955	53	COSMIC
COSN30167455	60	COSMIC
COSN30104028	115	COSMIC
COSN31567234	147	COSMIC
COSN20705502	156	COSMIC
COSN26676976	209	COSMIC
COSN21171288	243	COSMIC
COSN30130155	254	COSMIC
COSN31522137	315	COSMIC
COSN15010832	339	COSMIC
COSN28628592	357	COSMIC
COSN30173276	398	COSMIC
COSN31582668	413	COSMIC
COSN9297750	591	COSMIC
COSN31591732	634	COSMIC
COSN31584617	639	COSMIC
COSN31519872	670	COSMIC
COSN19662937	781	COSMIC
COSN31607670	827	COSMIC
COSN26644620	905	COSMIC
COSN31611299	927	COSMIC
COSN26554018	932	COSMIC
COSN2142575	997	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs926570043	4	dbSNP
rs375654397	6	dbSNP
rs1341276634	7	dbSNP
rs768135465	19	dbSNP
rs527777726	23	dbSNP
rs774837183	28	dbSNP
rs1323999181	32	dbSNP
rs766810780	33	dbSNP
rs182230788	34	dbSNP
rs558510756	36	dbSNP
rs746511000	38	dbSNP
rs1172460528	41	dbSNP
rs372156281	43	dbSNP
rs548281620	44	dbSNP
rs745855329	47	dbSNP
rs1322144828	48	dbSNP
rs769476003	51	dbSNP
rs951165129	54	dbSNP
rs866243756	57	dbSNP
rs1026686420	64	dbSNP
rs1206978945	65	dbSNP
rs995006558	73	dbSNP
rs967334970	74	dbSNP
rs148282184	77	dbSNP
rs899292635	84	dbSNP
rs1039702524	88	dbSNP
rs1182033370	89	dbSNP
rs1007782376	98	dbSNP
rs1181474693	116	dbSNP
rs1424791128	116	dbSNP
rs1362587151	117	dbSNP
rs890470553	122	dbSNP
rs992678632	127	dbSNP
rs1160552175	134	dbSNP
rs1404799126	140	dbSNP
rs970447958	142	dbSNP
rs1338326073	145	dbSNP
rs144017729	146	dbSNP
rs1002330467	154	dbSNP
rs56006088	156	dbSNP
rs761573548	166	dbSNP
rs776360099	170	dbSNP
rs1223157014	171	dbSNP
rs1304515411	173	dbSNP
rs945346138	173	dbSNP
rs1350026285	179	dbSNP
rs1280280998	185	dbSNP
rs1282533546	188	dbSNP
rs1322971385	189	dbSNP
rs546453106	194	dbSNP
rs1258007718	195	dbSNP
rs577499411	208	dbSNP
rs761389375	213	dbSNP
rs866641295	215	dbSNP
rs914022265	219	dbSNP
rs749944895	220	dbSNP
rs1016974376	223	dbSNP
rs1248947510	228	dbSNP
rs1421240435	228	dbSNP
rs936490503	233	dbSNP
rs926441602	234	dbSNP
rs981115948	235	dbSNP
rs1479181383	236	dbSNP
rs189186128	238	dbSNP
rs949356453	239	dbSNP
rs746884554	242	dbSNP
rs537173165	243	dbSNP
rs1296080619	244	dbSNP
rs1377658222	245	dbSNP
rs1316786173	246	dbSNP
rs567739003	247	dbSNP
rs540516618	249	dbSNP
rs574980491	252	dbSNP
rs901371834	253	dbSNP
rs770903591	254	dbSNP
rs149522218	262	dbSNP
rs544339812	263	dbSNP
rs1249817232	266	dbSNP
rs942475922	268	dbSNP
rs80155856	271	dbSNP
rs74813322	272	dbSNP
rs1206248234	276	dbSNP
rs925677394	277	dbSNP
rs576227178	278	dbSNP
rs1026694612	287	dbSNP
rs913156358	291	dbSNP
rs558615801	292	dbSNP
rs56326915	293	dbSNP
rs963894699	293	dbSNP
rs554671835	298	dbSNP
rs1430193001	301	dbSNP
rs1302242187	305	dbSNP
rs1363644697	306	dbSNP
rs756458732	307	dbSNP
rs538910302	311	dbSNP
rs752993672	315	dbSNP
rs376144722	318	dbSNP
rs1368463934	322	dbSNP
rs1381690097	323	dbSNP
rs984139030	324	dbSNP
rs951424133	326	dbSNP
rs55894848	329	dbSNP
rs1229600963	334	dbSNP
rs890742040	336	dbSNP
rs1030579997	338	dbSNP
rs536015632	341	dbSNP
rs901405867	356	dbSNP
rs1485278038	364	dbSNP
rs796452787	371	dbSNP
rs1335811343	372	dbSNP
rs1184210184	393	dbSNP
rs11554162	396	dbSNP
rs1018411759	397	dbSNP
rs1006990782	398	dbSNP
rs1162537196	399	dbSNP
rs904284270	411	dbSNP
rs1426294512	413	dbSNP
rs892431016	415	dbSNP
rs1344482191	417	dbSNP
rs945809868	427	dbSNP
rs1430117610	436	dbSNP
rs1206924391	438	dbSNP
rs567136692	447	dbSNP
rs1292832039	449	dbSNP
rs548247037	450	dbSNP
rs62627406	451	dbSNP
rs979949576	455	dbSNP
rs1213042723	465	dbSNP
rs1407763018	465	dbSNP
rs936739354	469	dbSNP
rs941801174	470	dbSNP
rs186276516	476	dbSNP
rs1357748854	488	dbSNP
rs180843823	490	dbSNP
rs1044936431	496	dbSNP
rs547421321	499	dbSNP
rs1398945010	501	dbSNP
rs951493598	508	dbSNP
rs949236564	511	dbSNP
rs976765705	514	dbSNP
rs917875235	516	dbSNP
rs1404116078	521	dbSNP
rs1165176861	523	dbSNP
rs79927819	524	dbSNP
rs188810026	525	dbSNP
rs929653261	527	dbSNP
rs775535692	528	dbSNP
rs763842021	529	dbSNP
rs1417088197	530	dbSNP
rs1185763843	534	dbSNP
rs1475631591	543	dbSNP
rs1329292932	546	dbSNP
rs1018444269	550	dbSNP
rs1260877218	569	dbSNP
rs1443716658	570	dbSNP
rs1210376993	582	dbSNP
rs1007021662	583	dbSNP
rs1232143498	583	dbSNP
rs904735039	587	dbSNP
rs919565678	591	dbSNP
rs7754021	592	dbSNP
rs1175730364	606	dbSNP
rs1010427665	610	dbSNP
rs759800888	611	dbSNP
rs1335090731	616	dbSNP
rs1354371150	624	dbSNP
rs1440433209	625	dbSNP
rs1310424478	627	dbSNP
rs865786709	633	dbSNP
rs1018427123	634	dbSNP
rs1272690483	639	dbSNP
rs1346487470	640	dbSNP
rs1214552195	643	dbSNP
rs1271811908	650	dbSNP
rs1435896282	656	dbSNP
rs1165802746	663	dbSNP
rs1337280566	669	dbSNP
rs986350031	670	dbSNP
rs1252468167	672	dbSNP
rs1271301396	673	dbSNP
rs1197188220	678	dbSNP
rs1214306328	683	dbSNP
rs1376053409	685	dbSNP
rs955042423	689	dbSNP
rs1479037855	690	dbSNP
rs1173418692	698	dbSNP
rs938335946	700	dbSNP
rs1428250751	703	dbSNP
rs1363093083	705	dbSNP
rs905779744	706	dbSNP
rs1030589452	708	dbSNP
rs1404938789	722	dbSNP
rs184076667	724	dbSNP
rs1399564170	725	dbSNP
rs1436521243	728	dbSNP
rs1269252265	736	dbSNP
rs532938039	743	dbSNP
rs1336978992	745	dbSNP
rs1044300082	752	dbSNP
rs1358449802	762	dbSNP
rs1009786686	773	dbSNP
rs892482209	774	dbSNP
rs1032318568	781	dbSNP
rs1326041057	781	dbSNP
rs530282561	782	dbSNP
rs1170699766	793	dbSNP
rs1391217645	793	dbSNP
rs1453434778	793	dbSNP
rs55984236	793	dbSNP
rs774779984	794	dbSNP
rs1169345359	795	dbSNP
rs1162010104	796	dbSNP
rs1410258739	798	dbSNP
rs1421407956	799	dbSNP
rs930079226	804	dbSNP
rs561489664	813	dbSNP
rs1316935054	815	dbSNP
rs918656910	816	dbSNP
rs977161536	818	dbSNP
rs965315688	819	dbSNP
rs58321558	828	dbSNP
rs1242291414	834	dbSNP
rs1267175211	835	dbSNP
rs1356895955	836	dbSNP
rs1045556306	841	dbSNP
rs1204471408	846	dbSNP
rs3836933	851	dbSNP
rs118055987	853	dbSNP
rs1283718295	853	dbSNP
rs75591900	855	dbSNP
rs1204103754	864	dbSNP
rs1248835616	865	dbSNP
rs544727374	868	dbSNP
rs1022241400	869	dbSNP
rs572802400	881	dbSNP
rs764749703	885	dbSNP
rs1473451180	894	dbSNP
rs1158483434	898	dbSNP
rs1385871358	905	dbSNP
rs552954115	907	dbSNP
rs1320850850	916	dbSNP
rs919824194	928	dbSNP
rs536567255	929	dbSNP
rs1038067168	934	dbSNP
rs942352430	935	dbSNP
rs1370546418	950	dbSNP
rs771278977	954	dbSNP
rs141074164	959	dbSNP
rs556770232	961	dbSNP
rs1210614803	967	dbSNP
rs1269021724	967	dbSNP
rs34857215	967	dbSNP
rs550125547	967	dbSNP
rs1006165586	976	dbSNP
rs1198941313	976	dbSNP
rs1258771163	977	dbSNP
rs887686797	978	dbSNP
rs1048034684	985	dbSNP
rs761413514	986	dbSNP
rs773172955	987	dbSNP
rs986913489	992	dbSNP
rs537936536	1002	dbSNP
rs1428174755	1013	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.