ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gagaucGCUCCUAAGGUUGUAa 5' :|| | ||||||| Target 5' tgggtcTGAACACCCCAACATc 3'	768 - 789	144.00	-8.80
2	miRNA 3' gaGAUCGCUCCUA-AGGUUGUAa 5' | ||| | || ||::|||| Target 5' gcCGAGCTTGCATGTCTGACATa 3'	235 - 257	128.00	-8.00
3	miRNA 3' gagaucgcuccuaagGUUGUAa 5' |||||| Target 5' atcatgtgcttaatgCAACATg 3'	555 - 576	120.00	-7.00

Mutant ID	Mutant Position	Mutant Source
COSN32067164	2	COSMIC
COSN20073532	16	COSMIC
COSN30171755	23	COSMIC
COSN31507564	24	COSMIC
COSN30465662	26	COSMIC
COSN30544799	36	COSMIC
COSN30449203	40	COSMIC
COSN30191239	54	COSMIC
COSN31556975	59	COSMIC
COSN30454914	65	COSMIC
COSN30169524	66	COSMIC
COSN31967776	66	COSMIC
COSN31967775	73	COSMIC
COSN8471434	84	COSMIC
COSN16130846	96	COSMIC
COSN30159131	112	COSMIC
COSN31576098	113	COSMIC
COSN30140406	123	COSMIC
COSN31505346	129	COSMIC
COSN30729739	131	COSMIC
COSN31526341	139	COSMIC
COSN30513372	140	COSMIC
COSN31599866	156	COSMIC
COSN5741647	157	COSMIC
COSN31967773	180	COSMIC
COSN31967772	181	COSMIC
COSN19565447	263	COSMIC
COSN6458245	274	COSMIC
COSN393385	449	COSMIC
COSN31967770	651	COSMIC
COSN31967769	672	COSMIC
COSN22852560	714	COSMIC
COSN31967768	740	COSMIC
COSN31967767	758	COSMIC
COSN8471433	791	COSMIC
COSN31967766	811	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs371275764	2	dbSNP
rs1234581902	3	dbSNP
rs1375228662	4	dbSNP
rs758411083	9	dbSNP
rs752621683	11	dbSNP
rs1462819196	12	dbSNP
rs1440901005	15	dbSNP
rs371480688	16	dbSNP
rs753544364	21	dbSNP
rs1413107770	22	dbSNP
rs1375233920	27	dbSNP
rs766104878	29	dbSNP
rs760231988	31	dbSNP
rs1378499907	33	dbSNP
rs1176664864	34	dbSNP
rs750003772	35	dbSNP
rs1182258201	36	dbSNP
rs772737025	36	dbSNP
rs946896473	38	dbSNP
rs900693133	39	dbSNP
rs763466642	42	dbSNP
rs775929602	44	dbSNP
rs944953368	45	dbSNP
rs770090187	46	dbSNP
rs912209394	49	dbSNP
rs1379979489	60	dbSNP
rs9333037	61	dbSNP
rs1432123561	63	dbSNP
rs370669636	64	dbSNP
rs1326282899	65	dbSNP
rs114160471	69	dbSNP
rs1264523613	77	dbSNP
rs1407425416	82	dbSNP
rs1198736498	84	dbSNP
rs1376350127	90	dbSNP
rs1366486225	91	dbSNP
rs1177432969	95	dbSNP
rs918337365	96	dbSNP
rs974271490	104	dbSNP
rs1443710547	106	dbSNP
rs962554653	110	dbSNP
rs1385731919	113	dbSNP
rs1386181988	116	dbSNP
rs1187126775	121	dbSNP
rs1347232426	127	dbSNP
rs1475703446	136	dbSNP
rs1326598922	141	dbSNP
rs916427561	141	dbSNP
rs1190819353	147	dbSNP
rs1242835435	148	dbSNP
rs535455348	150	dbSNP
rs990666488	152	dbSNP
rs1269281695	154	dbSNP
rs1221307212	160	dbSNP
rs1267309979	162	dbSNP
rs1200548898	168	dbSNP
rs59465754	170	dbSNP
rs1161512446	184	dbSNP
rs1457460481	189	dbSNP
rs1034944219	190	dbSNP
rs1214637150	195	dbSNP
rs1405369797	206	dbSNP
rs999490478	207	dbSNP
rs1339378023	212	dbSNP
rs1225180726	214	dbSNP
rs1307241739	218	dbSNP
rs1268917409	219	dbSNP
rs1439103651	223	dbSNP
rs1320602468	225	dbSNP
rs967146597	227	dbSNP
rs1022360743	230	dbSNP
rs555875183	234	dbSNP
rs537240596	237	dbSNP
rs569866933	238	dbSNP
rs1251593899	246	dbSNP
rs1479861460	247	dbSNP
rs1179232133	251	dbSNP
rs1251442616	259	dbSNP
rs878910482	263	dbSNP
rs1372570220	273	dbSNP
rs4660363	274	dbSNP
rs1425278621	275	dbSNP
rs551945063	275	dbSNP
rs1368537481	278	dbSNP
rs1444031385	280	dbSNP
rs1011437350	282	dbSNP
rs1377934805	286	dbSNP
rs1418764733	305	dbSNP
rs1166117042	306	dbSNP
rs1238154666	309	dbSNP
rs1481348390	317	dbSNP
rs1247431589	319	dbSNP
rs1274538119	330	dbSNP
rs1429750415	339	dbSNP
rs1198333226	342	dbSNP
rs1206983753	348	dbSNP
rs1483023010	356	dbSNP
rs1489756148	359	dbSNP
rs1445313714	360	dbSNP
rs1187864185	363	dbSNP
rs900642038	365	dbSNP
rs1234239671	368	dbSNP
rs1170821218	374	dbSNP
rs539782151	375	dbSNP
rs566344740	377	dbSNP
rs1436695584	378	dbSNP
rs1433639618	389	dbSNP
rs1270227959	390	dbSNP
rs1216715481	391	dbSNP
rs1225914988	394	dbSNP
rs547498637	395	dbSNP
rs1324828021	415	dbSNP
rs796431427	420	dbSNP
rs1009115719	422	dbSNP
rs1352624660	423	dbSNP
rs1240303665	427	dbSNP
rs1303292614	433	dbSNP
rs1476378933	437	dbSNP
rs1187004397	438	dbSNP
rs11211405	440	dbSNP
rs1441698320	443	dbSNP
rs1352857497	446	dbSNP
rs890721063	448	dbSNP
rs12735775	449	dbSNP
rs550253018	452	dbSNP
rs12735774	455	dbSNP
rs1346435012	456	dbSNP
rs1429898392	456	dbSNP
rs1433553621	457	dbSNP
rs1271283619	458	dbSNP
rs1175507785	463	dbSNP
rs1345342880	476	dbSNP
rs1479318893	481	dbSNP
rs1409767116	483	dbSNP
rs564167416	488	dbSNP
rs1282295918	490	dbSNP
rs11485679	509	dbSNP
rs12402176	523	dbSNP
rs1245997690	525	dbSNP
rs868717322	529	dbSNP
rs1201266649	537	dbSNP
rs1230694304	541	dbSNP
rs1204946348	542	dbSNP
rs9333045	557	dbSNP
rs9333046	558	dbSNP
rs796325731	559	dbSNP
rs1400773085	561	dbSNP
rs76535385	563	dbSNP
rs555842149	567	dbSNP
rs1403511067	568	dbSNP
rs1305387371	574	dbSNP
rs1346506195	575	dbSNP
rs1308091538	583	dbSNP
rs1223440875	584	dbSNP
rs537594338	587	dbSNP
rs1224345479	589	dbSNP
rs201817867	589	dbSNP
rs542994764	589	dbSNP
rs61784307	591	dbSNP
rs1482605672	592	dbSNP
rs558099833	592	dbSNP
rs1453450643	593	dbSNP
rs1470223790	594	dbSNP
rs746632544	619	dbSNP
rs1048043498	622	dbSNP
rs1406794912	626	dbSNP
rs1423338428	629	dbSNP
rs182771828	637	dbSNP
rs1468133896	644	dbSNP
rs1391122223	646	dbSNP
rs1327109625	647	dbSNP
rs1346771654	651	dbSNP
rs1159495963	652	dbSNP
rs1314520155	654	dbSNP
rs1399760924	655	dbSNP
rs143827915	656	dbSNP
rs1184963566	662	dbSNP
rs1444694881	668	dbSNP
rs918269469	672	dbSNP
rs1038108145	677	dbSNP
rs368226526	678	dbSNP
rs113060578	684	dbSNP
rs143758361	685	dbSNP
rs1186357821	686	dbSNP
rs1261680913	690	dbSNP
rs191716674	696	dbSNP
rs990615516	710	dbSNP
rs542775847	713	dbSNP
rs1294187643	720	dbSNP
rs9333051	724	dbSNP
rs9333052	726	dbSNP
rs1312955220	739	dbSNP
rs570903212	740	dbSNP
rs552108028	741	dbSNP
rs527446067	749	dbSNP
rs1363906995	752	dbSNP
rs1289301861	755	dbSNP
rs966723751	757	dbSNP
rs1325732833	758	dbSNP
rs1225122986	761	dbSNP
rs769155340	773	dbSNP
rs1343124198	774	dbSNP
rs9333053	777	dbSNP
rs9333054	778	dbSNP
rs1010969413	779	dbSNP
rs1260796465	790	dbSNP
rs529933832	792	dbSNP
rs1384086850	797	dbSNP
rs1203704217	802	dbSNP
rs1428493048	803	dbSNP
rs1161057504	805	dbSNP
rs1430872627	806	dbSNP
rs187780382	821	dbSNP
rs9333055	826	dbSNP
rs1009063245	836	dbSNP
rs1427383375	840	dbSNP
rs1471008176	847	dbSNP
rs1337521245	848	dbSNP
rs182628397	849	dbSNP
rs1485077838	851	dbSNP
rs1320189991	856	dbSNP
rs575301873	858	dbSNP
rs1223532270	864	dbSNP
rs1281011668	866	dbSNP
rs1167963711	873	dbSNP
rs1325185315	874	dbSNP
rs1223723594	880	dbSNP
rs1259674245	883	dbSNP
rs1484605709	886	dbSNP
rs12041693	897	dbSNP
rs1264405123	898	dbSNP
rs896786734	900	dbSNP
rs1038460603	901	dbSNP
rs941080034	903	dbSNP
rs916337997	907	dbSNP
rs1363353846	924	dbSNP
rs111621534	926	dbSNP
rs1160230505	928	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.