>YIPF2|NM_024029|3'UTR
   1 GAAGGCCCGGGTCCCACAGGCAACACCTAAGTGGACCAACCCCTCTGCCTGTCCTGCCCCCCAGACGATGACTGAAGGCT
  81 CCTTTGACACCTTGAGATGATTCTGCTACTTTCCAGACTTTTCTTACAAAGCAAACACTTTTATTTTCTATGCAAAGGTG
 161 ATTCAGAGAATTTATATAAAGGCGGGCGAGGGGCAGCCGAGCAGGGAGCTTTGGGACAGGGCTGGGGCCCCCATATCCCC
 241 CCCGGGCCACCTGCTTTCCCTCCTATGGCTCCCCTGGAACAGGAGGGAGAGCCAAGGGGGCGGCCCAGCCTGGACAGCGC
 321 CCGCTCCTGCCTGGGTGCACACACGGCGGGCCTGAGCTCCAGCATCTGAGTTTGGGGGTATGAGAAACAGGGGAGCAGAA
 401 GGAGAAGAAAACTGCCTGTGCTGCAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cuaCGGGACCGG----ACAGGGGCGu 5' ||: |||| | ||||| | Target 5' aggGCTGGGGCCCCCATATCCCCCCc 3'	218 - 243	121.00	-16.40
2	miRNA 3' cuACGGGACCGGAC--AGGGGcgu 5' | |||| : || ||||| Target 5' ctTTCCCTCCTATGGCTCCCCtgg 3'	254 - 277	114.00	-15.50
3	miRNA 3' cuACGGGACCGGACAGGGGcgu 5' || |||| |||| |||| Target 5' tcTG-CCTGTCCTGCCCCCcag 3'	44 - 64	107.00	-23.40

Mutant ID	Mutant Position	Mutant Source
COSN27005396	8	COSMIC
COSN5801985	163	COSMIC
COSN31595238	173	COSMIC
COSN1210616	180	COSMIC
COSN31594585	198	COSMIC
COSN31548934	265	COSMIC
COSN22419976	724	COSMIC
COSN26549064	856	COSMIC
COSN31489311	891	COSMIC
COSN31603509	948	COSMIC
COSN6137620	962	COSMIC
COSN28705941	964	COSMIC
COSN5849249	965	COSMIC
COSN31537074	971	COSMIC
COSN17443946	976	COSMIC
COSN30545304	982	COSMIC
COSN31584182	1027	COSMIC
COSN31519494	1067	COSMIC
COSN31609376	1084	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs755778567	7	dbSNP
rs752446208	8	dbSNP
rs376581863	9	dbSNP
rs1364706662	11	dbSNP
rs1410057204	13	dbSNP
rs562526305	15	dbSNP
rs766717626	19	dbSNP
rs751498636	20	dbSNP
rs766168799	24	dbSNP
rs762877735	25	dbSNP
rs1335652029	26	dbSNP
rs1323312314	30	dbSNP
rs368758760	34	dbSNP
rs202096110	35	dbSNP
rs1387653344	36	dbSNP
rs765070111	42	dbSNP
rs966210551	48	dbSNP
rs1019519339	50	dbSNP
rs761996468	53	dbSNP
rs776787935	54	dbSNP
rs768771346	56	dbSNP
rs573989449	57	dbSNP
rs113750118	59	dbSNP
rs1219148560	60	dbSNP
rs1490806675	62	dbSNP
rs775645969	65	dbSNP
rs1223058941	66	dbSNP
rs185388712	68	dbSNP
rs1341614009	73	dbSNP
rs534231570	75	dbSNP
rs554103980	76	dbSNP
rs763314802	81	dbSNP
rs1435990160	84	dbSNP
rs1274592729	91	dbSNP
rs1403535401	92	dbSNP
rs1367979374	94	dbSNP
rs891885832	97	dbSNP
rs776089584	102	dbSNP
rs771460764	114	dbSNP
rs1352195154	115	dbSNP
rs1225833177	116	dbSNP
rs747830345	117	dbSNP
rs780762189	122	dbSNP
rs1374422613	124	dbSNP
rs1378220915	130	dbSNP
rs754670257	130	dbSNP
rs1393719600	137	dbSNP
rs750963587	142	dbSNP
rs770106208	144	dbSNP
rs779566558	153	dbSNP
rs751074764	154	dbSNP
rs1281845188	157	dbSNP
rs1482033087	158	dbSNP
rs895756352	159	dbSNP
rs759864774	161	dbSNP
rs758313649	163	dbSNP
rs750404947	164	dbSNP
rs1037508546	166	dbSNP
rs1490183162	170	dbSNP
rs765014949	171	dbSNP
rs761657336	175	dbSNP
rs1219973313	177	dbSNP
rs1347857356	181	dbSNP
rs1465697980	185	dbSNP
rs1263642277	186	dbSNP
rs1284331462	186	dbSNP
rs939891141	189	dbSNP
rs573342385	199	dbSNP
rs1376136851	205	dbSNP
rs909602814	209	dbSNP
rs192517427	211	dbSNP
rs940139720	214	dbSNP
rs760989205	215	dbSNP
rs1441301432	218	dbSNP
rs1163728665	219	dbSNP
rs1380754117	220	dbSNP
rs1386403447	221	dbSNP
rs763979510	223	dbSNP
rs1462085386	231	dbSNP
rs1358192406	233	dbSNP
rs1387629498	234	dbSNP
rs539486417	248	dbSNP
rs1317914779	252	dbSNP
rs932297853	255	dbSNP
rs1248247569	257	dbSNP
rs569010084	259	dbSNP
rs1358613415	261	dbSNP
rs1243711921	272	dbSNP
rs976475937	274	dbSNP
rs932531915	279	dbSNP
rs187656203	280	dbSNP
rs1019235370	281	dbSNP
rs182980730	284	dbSNP
rs568353844	285	dbSNP
rs546826138	289	dbSNP
rs1441897499	291	dbSNP
rs867079738	294	dbSNP
rs752684657	295	dbSNP
rs1033068961	296	dbSNP
rs1318873468	302	dbSNP
rs1454644989	303	dbSNP
rs1034643773	305	dbSNP
rs1386723880	309	dbSNP
rs1000292949	315	dbSNP
rs1337413482	324	dbSNP
rs1382164871	329	dbSNP
rs1394149681	331	dbSNP
rs528397604	333	dbSNP
rs1023654395	334	dbSNP
rs534105133	337	dbSNP
rs371478391	338	dbSNP
rs895714221	340	dbSNP
rs765052900	341	dbSNP
rs1037237592	347	dbSNP
rs1180564377	356	dbSNP
rs1253271966	357	dbSNP
rs1420275856	362	dbSNP
rs1448436394	364	dbSNP
rs1245834870	369	dbSNP
rs552120292	372	dbSNP
rs377766404	374	dbSNP
rs1004253171	386	dbSNP
rs533228858	388	dbSNP
rs375336858	398	dbSNP
rs539579693	398	dbSNP
rs1036918323	399	dbSNP
rs1003963414	407	dbSNP
rs892699177	415	dbSNP
rs1286175770	416	dbSNP
rs1347200376	417	dbSNP
rs921168298	418	dbSNP
rs562687912	419	dbSNP
rs879151285	420	dbSNP
rs1282261689	421	dbSNP
rs544188745	423	dbSNP
rs932382078	428	dbSNP
rs1251426633	429	dbSNP
rs1249841191	432	dbSNP
rs1235119585	434	dbSNP
rs762660495	436	dbSNP
rs543145800	439	dbSNP
rs191874989	441	dbSNP
rs561944486	442	dbSNP
rs912088811	444	dbSNP
rs540896463	445	dbSNP
rs989078384	448	dbSNP
rs1394798440	451	dbSNP
rs956162254	454	dbSNP
rs868137064	457	dbSNP
rs987974945	460	dbSNP
rs960409329	464	dbSNP
rs1306116788	468	dbSNP
rs1173656600	472	dbSNP
rs752758657	476	dbSNP
rs978948294	486	dbSNP
rs1387898533	487	dbSNP
rs980408167	487	dbSNP
rs769310688	488	dbSNP
rs1365014181	490	dbSNP
rs1027354360	496	dbSNP
rs974724418	499	dbSNP
rs570247069	502	dbSNP
rs970543684	506	dbSNP
rs1273091646	516	dbSNP
rs187876258	516	dbSNP
rs558317096	524	dbSNP
rs1014637318	526	dbSNP
rs546448252	527	dbSNP
rs1487598609	532	dbSNP
rs1188540901	533	dbSNP
rs1420234121	539	dbSNP
rs548222142	546	dbSNP
rs1428010320	548	dbSNP
rs1166856017	550	dbSNP
rs1387014279	551	dbSNP
rs754664127	554	dbSNP
rs1302284210	556	dbSNP
rs960071774	556	dbSNP
rs1015442686	561	dbSNP
rs1289097342	564	dbSNP
rs1347926657	564	dbSNP
rs1432371088	564	dbSNP
rs1258154168	565	dbSNP
rs1432253251	566	dbSNP
rs1004100577	567	dbSNP
rs1362191948	572	dbSNP
rs891181999	572	dbSNP
rs1029721148	578	dbSNP
rs996915413	581	dbSNP
rs1320300418	585	dbSNP
rs747612239	589	dbSNP
rs899823057	591	dbSNP
rs1272635524	595	dbSNP
rs1048198679	597	dbSNP
rs996732833	599	dbSNP
rs1043772620	602	dbSNP
rs1186725510	603	dbSNP
rs1245096638	609	dbSNP
rs575408639	626	dbSNP
rs1040799059	627	dbSNP
rs1442764227	629	dbSNP
rs1402216759	632	dbSNP
rs1243090967	639	dbSNP
rs1032952349	649	dbSNP
rs939182822	658	dbSNP
rs913582016	661	dbSNP
rs1314060762	663	dbSNP
rs1409787317	667	dbSNP
rs1052128254	674	dbSNP
rs1368132294	676	dbSNP
rs1284141547	679	dbSNP
rs774591824	685	dbSNP
rs1225827621	689	dbSNP
rs980506379	693	dbSNP
rs926231302	695	dbSNP
rs868363319	698	dbSNP
rs979331529	699	dbSNP
rs1207088065	703	dbSNP
rs557134503	706	dbSNP
rs372564981	707	dbSNP
rs368879821	708	dbSNP
rs992945083	713	dbSNP
rs960285123	714	dbSNP
rs535554136	715	dbSNP
rs568019879	721	dbSNP
rs1177546390	723	dbSNP
rs1407006095	724	dbSNP
rs1418435076	725	dbSNP
rs1181336878	742	dbSNP
rs952620546	744	dbSNP
rs1015812595	745	dbSNP
rs1331706041	747	dbSNP
rs982891072	748	dbSNP
rs1445056421	749	dbSNP
rs552708981	755	dbSNP
rs997042006	756	dbSNP
rs1226736144	757	dbSNP
rs1285412405	758	dbSNP
rs1324619130	763	dbSNP
rs1289462685	765	dbSNP
rs899679940	773	dbSNP
rs1439439087	774	dbSNP
rs1205172567	777	dbSNP
rs1250710510	783	dbSNP
rs1019760659	785	dbSNP
rs938661126	786	dbSNP
rs1201392002	796	dbSNP
rs1429549038	798	dbSNP
rs1421057553	802	dbSNP
rs199572333	802	dbSNP
rs899916252	802	dbSNP
rs1412096330	803	dbSNP
rs768522571	804	dbSNP
rs1228301360	806	dbSNP
rs1349251871	807	dbSNP
rs1280718832	808	dbSNP
rs1370541577	808	dbSNP
rs1408379690	809	dbSNP
rs1442315826	810	dbSNP
rs1000980474	812	dbSNP
rs534901384	813	dbSNP
rs1341565667	814	dbSNP
rs892251011	818	dbSNP
rs1273813103	819	dbSNP
rs182904636	820	dbSNP
rs892362562	820	dbSNP
rs1448380060	821	dbSNP
rs1196347434	824	dbSNP
rs1243279388	826	dbSNP
rs939275990	827	dbSNP
rs1390062899	828	dbSNP
rs1165808870	829	dbSNP
rs373412970	829	dbSNP
rs1158237155	832	dbSNP
rs1455516267	835	dbSNP
rs1386361010	836	dbSNP
rs1321843195	837	dbSNP
rs376292509	838	dbSNP
rs1431568947	841	dbSNP
rs1044960234	842	dbSNP
rs936361528	843	dbSNP
rs920366501	856	dbSNP
rs1246244288	861	dbSNP
rs904737438	873	dbSNP
rs1043322333	874	dbSNP
rs907569979	879	dbSNP
rs1447259437	880	dbSNP
rs552344323	888	dbSNP
rs1208495265	891	dbSNP
rs955661202	895	dbSNP
rs948871163	900	dbSNP
rs1369352659	901	dbSNP
rs747054463	904	dbSNP
rs1161899663	906	dbSNP
rs1327627290	920	dbSNP
rs916099399	923	dbSNP
rs1387378112	924	dbSNP
rs1458011479	930	dbSNP
rs993427189	933	dbSNP
rs749779974	939	dbSNP
rs1363161601	944	dbSNP
rs1454447767	945	dbSNP
rs922732837	947	dbSNP
rs975613971	950	dbSNP
rs938753350	952	dbSNP
rs964445082	956	dbSNP
rs12972875	961	dbSNP
rs569520938	962	dbSNP
rs190617590	964	dbSNP
rs12974658	965	dbSNP
rs1026832236	967	dbSNP
rs1045089749	971	dbSNP
rs12972716	973	dbSNP
rs12972709	980	dbSNP
rs529371765	981	dbSNP
rs1019432776	982	dbSNP
rs562082626	983	dbSNP
rs892218459	985	dbSNP
rs1030759810	986	dbSNP
rs1455692995	990	dbSNP
rs1470991144	992	dbSNP
rs540304761	994	dbSNP
rs369706119	1002	dbSNP
rs1394238989	1003	dbSNP
rs11543971	1005	dbSNP
rs1308821957	1006	dbSNP
rs907546369	1008	dbSNP
rs1188269363	1009	dbSNP
rs1000499554	1011	dbSNP
rs903534469	1013	dbSNP
rs528762842	1018	dbSNP
rs564674040	1019	dbSNP
rs894746948	1023	dbSNP
rs964224453	1024	dbSNP
rs1253021760	1026	dbSNP
rs546411343	1027	dbSNP
rs1277487487	1028	dbSNP
rs1481568316	1032	dbSNP
rs575866523	1051	dbSNP
rs11543972	1052	dbSNP
rs1030968916	1056	dbSNP
rs557591642	1058	dbSNP
rs1299828747	1059	dbSNP
rs541911605	1061	dbSNP
rs970605427	1062	dbSNP
rs1023572684	1064	dbSNP
rs1346984078	1072	dbSNP
rs931293269	1075	dbSNP
rs1219471040	1077	dbSNP
rs1432496610	1082	dbSNP
rs1363785413	1085	dbSNP
rs1012601146	1093	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.