ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uaGACGGACUCGUAGAGGaa 5' :| |: || |||||| Target 5' taTTACTAAAGAATCTCCcc 3'	381 - 400	130.00	-8.30
2	miRNA 3' uagacggacucGUAGAGGAa 5' :||||:|| Target 5' ctagatttttcTATCTTCTg 3'	485 - 504	125.00	-6.50
3	miRNA 3' uagacggacucguAGAGGAa 5' |||||| Target 5' acaggatctcagtTCTCCTt 3'	89 - 108	120.00	-12.10

Mutant ID	Mutant Position	Mutant Source
COSN505148	7	COSMIC
COSN1852869	13	COSMIC
COSN26553350	19	COSMIC
COSN30520431	19	COSMIC
COSN2375766	31	COSMIC
COSN30140202	32	COSMIC
COSN31971405	33	COSMIC
COSN30490663	34	COSMIC
COSN30479141	35	COSMIC
COSN31583359	111	COSMIC
COSN14004621	126	COSMIC
COSN31558037	129	COSMIC
COSN18726917	161	COSMIC
COSN31524985	212	COSMIC
COSN1241953	226	COSMIC
COSN30126376	303	COSMIC
COSN27508995	421	COSMIC
COSN9385206	436	COSMIC
COSN25786935	524	COSMIC
COSN29074741	531	COSMIC
COSN27722846	582	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs763971515	5	dbSNP
rs751255650	6	dbSNP
rs770484335	7	dbSNP
rs1300613918	10	dbSNP
rs780986690	19	dbSNP
rs745425927	20	dbSNP
rs1165489191	23	dbSNP
rs752687200	23	dbSNP
rs755916085	23	dbSNP
rs79372020	24	dbSNP
rs755660423	26	dbSNP
rs779888472	33	dbSNP
rs748983050	37	dbSNP
rs1297997415	38	dbSNP
rs1344619787	40	dbSNP
rs868398384	43	dbSNP
rs768148626	44	dbSNP
rs1211426969	45	dbSNP
rs774794901	46	dbSNP
rs539578281	56	dbSNP
rs925730772	62	dbSNP
rs1202184760	63	dbSNP
rs556184634	64	dbSNP
rs957016726	66	dbSNP
rs1189651167	75	dbSNP
rs772704468	80	dbSNP
rs760753173	84	dbSNP
rs1429985328	87	dbSNP
rs773580515	90	dbSNP
rs760792826	91	dbSNP
rs1376638539	93	dbSNP
rs777698368	103	dbSNP
rs1338053699	104	dbSNP
rs114879649	106	dbSNP
rs749104008	107	dbSNP
rs756901863	113	dbSNP
rs541499519	114	dbSNP
rs936668514	116	dbSNP
rs759766805	120	dbSNP
rs561650714	126	dbSNP
rs1046091549	129	dbSNP
rs180689187	130	dbSNP
rs1284462214	132	dbSNP
rs868049761	135	dbSNP
rs1459751767	140	dbSNP
rs1048130	145	dbSNP
rs1159017174	148	dbSNP
rs564137993	149	dbSNP
rs1047136785	157	dbSNP
rs888497437	163	dbSNP
rs532606939	167	dbSNP
rs1005595490	168	dbSNP
rs765466464	172	dbSNP
rs1453383099	173	dbSNP
rs932462285	208	dbSNP
rs150181431	211	dbSNP
rs1204419365	212	dbSNP
rs1039655898	213	dbSNP
rs1259987210	219	dbSNP
rs371591908	224	dbSNP
rs71397377	224	dbSNP
rs746063532	224	dbSNP
rs1038957203	225	dbSNP
rs749633112	226	dbSNP
rs775626982	226	dbSNP
rs867609197	234	dbSNP
rs897349802	235	dbSNP
rs994233165	240	dbSNP
rs1030349607	258	dbSNP
rs1232206168	263	dbSNP
rs903347287	269	dbSNP
rs1302210565	271	dbSNP
rs999461540	280	dbSNP
rs1388815662	289	dbSNP
rs1033183671	303	dbSNP
rs1187976708	310	dbSNP
rs1360301937	315	dbSNP
rs1297992701	317	dbSNP
rs1420773051	331	dbSNP
rs1004044998	339	dbSNP
rs957087983	344	dbSNP
rs1474348528	347	dbSNP
rs1161472643	350	dbSNP
rs1048139	352	dbSNP
rs532024600	355	dbSNP
rs1377763814	368	dbSNP
rs1200531960	387	dbSNP
rs376798669	389	dbSNP
rs995207644	400	dbSNP
rs1033836142	411	dbSNP
rs11540989	411	dbSNP
rs959186610	411	dbSNP
rs77691716	412	dbSNP
rs1287586277	415	dbSNP
rs1014168874	419	dbSNP
rs1256728072	421	dbSNP
rs1344397699	421	dbSNP
rs1404936564	422	dbSNP
rs1397024259	454	dbSNP
rs1305989391	459	dbSNP
rs971076198	462	dbSNP
rs891122424	474	dbSNP
rs1342832651	478	dbSNP
rs1401017134	478	dbSNP
rs1311585713	489	dbSNP
rs1396018504	496	dbSNP
rs779386283	505	dbSNP
rs186647495	506	dbSNP
rs10198316	510	dbSNP
rs1231003086	511	dbSNP
rs1422495538	514	dbSNP
rs1171005786	515	dbSNP
rs1380856933	515	dbSNP
rs200606303	515	dbSNP
rs1256877193	517	dbSNP
rs569717963	521	dbSNP
rs925543251	521	dbSNP
rs1289693007	522	dbSNP
rs958005434	522	dbSNP
rs533895933	523	dbSNP
rs1295353949	524	dbSNP
rs1331602063	524	dbSNP
rs547706758	524	dbSNP
rs1048165	525	dbSNP
rs1258943277	526	dbSNP
rs1473062640	530	dbSNP
rs922002189	531	dbSNP
rs1179638728	533	dbSNP
rs1417368245	534	dbSNP
rs933457733	539	dbSNP
rs1199442005	540	dbSNP
rs539301660	543	dbSNP
rs1271615982	547	dbSNP
rs776867919	551	dbSNP
rs939877978	552	dbSNP
rs1184559520	557	dbSNP
rs1286433838	572	dbSNP
rs1036892404	574	dbSNP
rs1412840398	574	dbSNP
rs1309374846	579	dbSNP
rs981379784	583	dbSNP
rs556313077	585	dbSNP
rs1405717290	588	dbSNP
rs759791275	589	dbSNP
rs1392669822	596	dbSNP
rs763852311	600	dbSNP
rs995573286	620	dbSNP
rs1419032734	621	dbSNP
rs189430193	622	dbSNP
rs1048195	623	dbSNP
rs1179372457	631	dbSNP
rs894807218	635	dbSNP
rs1172364212	650	dbSNP
rs1013302614	651	dbSNP
rs1198468863	654	dbSNP
rs72904951	658	dbSNP
rs1292540056	659	dbSNP
rs182117922	664	dbSNP
rs1353628932	665	dbSNP
rs1286065428	669	dbSNP
rs1350704636	681	dbSNP
rs1390377018	682	dbSNP
rs941419210	683	dbSNP
rs1039791286	697	dbSNP
rs1436839547	719	dbSNP
rs899825273	720	dbSNP
rs1279376404	729	dbSNP
rs572088227	737	dbSNP
rs1345424741	742	dbSNP
rs1206990237	751	dbSNP
rs934023073	753	dbSNP
rs1361115024	758	dbSNP
rs1160431411	765	dbSNP
rs1252867910	770	dbSNP
rs1423231539	783	dbSNP
rs1480421276	788	dbSNP
rs1175620106	790	dbSNP
rs1478488381	796	dbSNP
rs1248753330	800	dbSNP
rs1191991899	814	dbSNP
rs1051343269	831	dbSNP
rs186008502	836	dbSNP
rs1219489427	841	dbSNP
rs999117119	843	dbSNP
rs1193506302	845	dbSNP
rs1277374961	852	dbSNP
rs1028952138	853	dbSNP
rs1346718271	862	dbSNP
rs1268143409	865	dbSNP
rs1393690472	867	dbSNP
rs201568624	869	dbSNP
rs987552432	878	dbSNP
rs893340220	881	dbSNP
rs1412918717	883	dbSNP
rs907992167	884	dbSNP
rs1333600077	895	dbSNP
rs1461114093	896	dbSNP
rs1415217696	907	dbSNP
rs138728342	910	dbSNP
rs1477684121	915	dbSNP
rs1424876471	916	dbSNP
rs563084193	923	dbSNP
rs1022760748	925	dbSNP
rs971079792	929	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.