ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cggacguaGUACUUUACGGGAGc 5' ||| ||||||| Target 5' gccgtcccCATCCCCTGCCCTCa 3'	613 - 635	143.00	-15.40
2	miRNA 3' cgGACGUAGUAC----UUU-ACGGGagc 5' |||| || || ||| ||||| Target 5' ccCTGCCTCTTGTCACAAACTGCCCcag 3'	693 - 720	127.00	-15.90
3	miRNA 3' cgGACGU-AGUACUUUACGGGAGc 5' ||||| ||||| |||:|| Target 5' gcCTGCACACATGA---GCCTTCc 3'	102 - 122	125.00	-20.90

Mutant ID	Mutant Position	Mutant Source
COSN23017098	20	COSMIC
COSN31484227	35	COSMIC
COSN30521555	45	COSMIC
COSN31564800	54	COSMIC
COSN14300312	62	COSMIC
COSN30757988	66	COSMIC
COSN24310900	115	COSMIC
COSN15262820	159	COSMIC
COSN20076071	175	COSMIC
COSN5103236	202	COSMIC
COSN6891278	397	COSMIC
COSN17075729	405	COSMIC
COSN9770767	520	COSMIC
rs188982745	412	GWAS

Mutant ID	Mutant Position	Mutant Source
rs782715921	5	dbSNP
rs781802427	6	dbSNP
rs782474413	7	dbSNP
rs373582004	9	dbSNP
rs782140570	10	dbSNP
rs373913398	17	dbSNP
rs782039518	18	dbSNP
rs370470463	19	dbSNP
rs782342027	20	dbSNP
rs375576005	22	dbSNP
rs1368160848	24	dbSNP
rs1471913558	27	dbSNP
rs781978459	28	dbSNP
rs782388247	30	dbSNP
rs782292155	31	dbSNP
rs782465879	33	dbSNP
rs782738627	37	dbSNP
rs782209775	45	dbSNP
rs1377760533	50	dbSNP
rs1042700476	55	dbSNP
rs574800772	58	dbSNP
rs947058929	61	dbSNP
rs1334224097	62	dbSNP
rs1339548419	63	dbSNP
rs552864297	65	dbSNP
rs868932043	66	dbSNP
rs1354153192	67	dbSNP
rs534849362	68	dbSNP
rs1239109521	69	dbSNP
rs1283826093	70	dbSNP
rs1349931967	76	dbSNP
rs748613410	83	dbSNP
rs782185455	89	dbSNP
rs1211731500	97	dbSNP
rs1255551253	106	dbSNP
rs991392816	112	dbSNP
rs1184269254	114	dbSNP
rs938511475	114	dbSNP
rs1439509375	116	dbSNP
rs138977022	122	dbSNP
rs191652486	123	dbSNP
rs962121678	128	dbSNP
rs1016776522	138	dbSNP
rs536648176	139	dbSNP
rs953528752	148	dbSNP
rs1393722957	152	dbSNP
rs1029239264	158	dbSNP
rs1377118541	162	dbSNP
rs997804790	168	dbSNP
rs1445832653	174	dbSNP
rs1306764827	177	dbSNP
rs1373182599	183	dbSNP
rs80056196	187	dbSNP
rs113146366	189	dbSNP
rs776990992	189	dbSNP
rs769255998	190	dbSNP
rs745352851	193	dbSNP
rs778427893	194	dbSNP
rs756856634	195	dbSNP
rs1197593456	196	dbSNP
rs1258977221	197	dbSNP
rs1457467078	198	dbSNP
rs748955585	199	dbSNP
rs777561329	201	dbSNP
rs755853719	203	dbSNP
rs1000115740	207	dbSNP
rs1455131185	210	dbSNP
rs902908461	211	dbSNP
rs1198780778	226	dbSNP
rs1376386808	227	dbSNP
rs146117610	249	dbSNP
rs752592157	250	dbSNP
rs1400623156	259	dbSNP
rs1464773262	264	dbSNP
rs530298094	265	dbSNP
rs947058482	270	dbSNP
rs1300549457	274	dbSNP
rs868939280	275	dbSNP
rs1329231355	287	dbSNP
rs1229772330	288	dbSNP
rs1278887766	293	dbSNP
rs1317593793	298	dbSNP
rs1196971794	299	dbSNP
rs1275221663	305	dbSNP
rs782237582	309	dbSNP
rs1209563539	311	dbSNP
rs1265941803	314	dbSNP
rs1430776587	320	dbSNP
rs1194779520	331	dbSNP
rs782629533	332	dbSNP
rs1477175140	335	dbSNP
rs566387948	341	dbSNP
rs111685234	345	dbSNP
rs1292712483	345	dbSNP
rs1388541507	353	dbSNP
rs1383848742	354	dbSNP
rs1298945278	355	dbSNP
rs1339961103	356	dbSNP
rs1216830448	359	dbSNP
rs894167424	366	dbSNP
rs1295371602	367	dbSNP
rs1322531490	368	dbSNP
rs782514905	371	dbSNP
rs371070414	372	dbSNP
rs782682936	397	dbSNP
rs1218002182	398	dbSNP
rs1263012096	398	dbSNP
rs782431099	407	dbSNP
rs1487695750	410	dbSNP
rs188982745	412	dbSNP
rs1415577558	414	dbSNP
rs928563150	415	dbSNP
rs971845112	418	dbSNP
rs532780957	422	dbSNP
rs2237278	424	dbSNP
rs1359763500	431	dbSNP
rs1398277930	432	dbSNP
rs543889900	439	dbSNP
rs530643475	445	dbSNP
rs561234143	448	dbSNP
rs1319108489	455	dbSNP
rs1028957369	458	dbSNP
rs561646706	459	dbSNP
rs184186065	474	dbSNP
rs1213877182	489	dbSNP
rs1238701153	497	dbSNP
rs1483408649	512	dbSNP
rs1180940897	517	dbSNP
rs1253563127	518	dbSNP
rs1441335466	519	dbSNP
rs751647516	520	dbSNP
rs766588271	522	dbSNP
rs1379653530	528	dbSNP
rs1417778262	532	dbSNP
rs35107030	533	dbSNP
rs1359829076	534	dbSNP
rs1465549240	545	dbSNP
rs1301347661	554	dbSNP
rs1375740292	556	dbSNP
rs773571989	557	dbSNP
rs1448048247	563	dbSNP
rs1308950419	576	dbSNP
rs1375418022	577	dbSNP
rs80016682	579	dbSNP
rs1279888212	580	dbSNP
rs1312272989	586	dbSNP
rs34329931	590	dbSNP
rs1253927807	596	dbSNP
rs1179090053	597	dbSNP
rs1458791351	597	dbSNP
rs1237323357	603	dbSNP
rs1437528070	605	dbSNP
rs1192854432	606	dbSNP
rs1425735874	612	dbSNP
rs370717617	613	dbSNP
rs1414000863	615	dbSNP
rs540862201	616	dbSNP
rs1402782008	622	dbSNP
rs1466334587	622	dbSNP
rs1331169404	625	dbSNP
rs577016467	626	dbSNP
rs1011782161	627	dbSNP
rs1443484308	638	dbSNP
rs558336322	643	dbSNP
rs1367485766	644	dbSNP
rs1218169530	645	dbSNP
rs1298549250	650	dbSNP
rs536712718	651	dbSNP
rs376624403	654	dbSNP
rs782734578	655	dbSNP
rs782085942	656	dbSNP
rs1256953767	664	dbSNP
rs781974766	667	dbSNP
rs1223432217	669	dbSNP
rs1247271164	678	dbSNP
rs575753139	686	dbSNP
rs1036713993	694	dbSNP
rs1189615304	701	dbSNP
rs1425856222	704	dbSNP
rs940524806	704	dbSNP
rs1421709543	716	dbSNP
rs1407463002	721	dbSNP
rs1324580703	724	dbSNP
rs782372480	733	dbSNP
rs1349874237	736	dbSNP
rs573569060	741	dbSNP
rs1434924693	743	dbSNP
rs1300520830	745	dbSNP
rs1341840069	749	dbSNP
rs782259609	750	dbSNP
rs782015318	756	dbSNP
rs932022772	757	dbSNP
rs782416124	761	dbSNP
rs1229863149	767	dbSNP
rs1451024773	769	dbSNP
rs922022271	769	dbSNP
rs1219302193	774	dbSNP
rs1265167299	777	dbSNP
rs1488677968	802	dbSNP
rs976057559	806	dbSNP
rs1261349737	815	dbSNP
rs966058666	824	dbSNP
rs1164254639	827	dbSNP
rs553676252	862	dbSNP
rs1403887552	900	dbSNP
rs1045200	902	dbSNP
rs1161005783	915	dbSNP
rs1361677437	928	dbSNP
rs1400129629	929	dbSNP
rs1297010397	931	dbSNP
rs62466282	931	dbSNP
rs1449751287	932	dbSNP
rs1284366154	941	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.