ID	Duplex structure	Position	Score	MFE
1	miRNA 3' acCUGCAAACGUC-CCCUCCACc 5' ||| || | || ||||||| Target 5' agGAC-TTGGGAGCTGGAGGTGt 3'	946 - 967	155.00	-18.80
2	miRNA 3' acCUG----CAAACG--UCC-CCUCCAcc 5' ||| ||| |: ||| |||||| Target 5' ggGACATGGGTTAGTTAAGGCGGAGGTac 3'	12 - 40	136.00	-17.40
3	miRNA 3' acCUGCAAACGUCCCCUCCACc 5' |:| || ||| ||||| | Target 5' tgGGCACTT-CAGAGGAGGAGc 3'	353 - 373	127.00	-16.42

Mutant ID	Mutant Position	Mutant Source
COSN27006560	40	COSMIC
COSN30447575	46	COSMIC
COSN30188841	52	COSMIC
COSN27006559	54	COSMIC
COSN27006561	63	COSMIC
COSN19718608	68	COSMIC
COSN30499602	77	COSMIC
COSN31488097	80	COSMIC
COSN30526289	94	COSMIC
COSN31526839	298	COSMIC
COSN31553362	350	COSMIC
COSN31564941	380	COSMIC
COSN30163639	572	COSMIC
COSN31580080	627	COSMIC
COSN31539234	645	COSMIC
COSN31548949	829	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs759334092	1	dbSNP
rs202050969	9	dbSNP
rs929657211	16	dbSNP
rs1410338022	17	dbSNP
rs1402839633	18	dbSNP
rs770808170	19	dbSNP
rs576654285	20	dbSNP
rs754338641	29	dbSNP
rs368446487	32	dbSNP
rs1368775764	33	dbSNP
rs766855100	38	dbSNP
rs771691691	40	dbSNP
rs369445815	41	dbSNP
rs754710921	42	dbSNP
rs750754036	43	dbSNP
rs781392516	45	dbSNP
rs752006821	46	dbSNP
rs764644099	47	dbSNP
rs1266383044	48	dbSNP
rs375884266	49	dbSNP
rs1352148529	51	dbSNP
rs1214212237	56	dbSNP
rs1449214106	63	dbSNP
rs911154017	66	dbSNP
rs1245694413	68	dbSNP
rs1471411808	69	dbSNP
rs1159570476	70	dbSNP
rs985229645	71	dbSNP
rs186276397	76	dbSNP
rs1357759408	81	dbSNP
rs1312377451	83	dbSNP
rs1175732102	91	dbSNP
rs1247757799	98	dbSNP
rs1409317374	99	dbSNP
rs1306182029	103	dbSNP
rs1352271080	110	dbSNP
rs1442818260	110	dbSNP
rs755597763	111	dbSNP
rs1379201470	113	dbSNP
rs1269059727	117	dbSNP
rs955391535	118	dbSNP
rs922671676	124	dbSNP
rs146313245	132	dbSNP
rs1340940236	138	dbSNP
rs544987257	139	dbSNP
rs573061134	142	dbSNP
rs1010930673	145	dbSNP
rs959583640	150	dbSNP
rs1246538495	163	dbSNP
rs73615044	165	dbSNP
rs536075062	167	dbSNP
rs1003676749	172	dbSNP
rs1166854862	190	dbSNP
rs906735537	191	dbSNP
rs1045480169	195	dbSNP
rs1419135551	199	dbSNP
rs1322839661	200	dbSNP
rs77029730	218	dbSNP
rs1364090784	226	dbSNP
rs1476469493	226	dbSNP
rs1243184586	229	dbSNP
rs964022373	231	dbSNP
rs1299228228	243	dbSNP
rs763194196	244	dbSNP
rs1222408864	245	dbSNP
rs554692852	250	dbSNP
rs537903812	252	dbSNP
rs1008792810	256	dbSNP
rs911104896	257	dbSNP
rs1049635671	258	dbSNP
rs569171016	261	dbSNP
rs922462026	265	dbSNP
rs890305221	276	dbSNP
rs552510050	277	dbSNP
rs993727499	280	dbSNP
rs898951711	282	dbSNP
rs182130125	296	dbSNP
rs768438165	297	dbSNP
rs937706727	305	dbSNP
rs926263093	307	dbSNP
rs1363465794	308	dbSNP
rs1046647549	314	dbSNP
rs1432514786	324	dbSNP
rs1297302066	326	dbSNP
rs1278685860	334	dbSNP
rs1326609140	340	dbSNP
rs915341212	343	dbSNP
rs989445892	366	dbSNP
rs538985867	373	dbSNP
rs1274240093	377	dbSNP
rs567182427	380	dbSNP
rs959361594	390	dbSNP
rs1343352773	393	dbSNP
rs191033930	394	dbSNP
rs186134244	399	dbSNP
rs1366797527	400	dbSNP
rs914065803	401	dbSNP
rs970905137	402	dbSNP
rs1411083288	403	dbSNP
rs775398247	409	dbSNP
rs561419454	411	dbSNP
rs988361690	413	dbSNP
rs958444647	423	dbSNP
rs1395072791	424	dbSNP
rs1331704430	425	dbSNP
rs1168673589	429	dbSNP
rs550703318	433	dbSNP
rs759344629	434	dbSNP
rs975483527	435	dbSNP
rs964055661	438	dbSNP
rs1479730837	440	dbSNP
rs1271735591	442	dbSNP
rs530594206	449	dbSNP
rs751729	456	dbSNP
rs1009234232	457	dbSNP
rs1351756919	462	dbSNP
rs745364963	464	dbSNP
rs1026645442	477	dbSNP
rs138159244	484	dbSNP
rs545014358	487	dbSNP
rs778438029	492	dbSNP
rs898979085	493	dbSNP
rs1250991099	496	dbSNP
rs866712847	508	dbSNP
rs1471533465	516	dbSNP
rs1161087640	522	dbSNP
rs1222420162	522	dbSNP
rs756751718	523	dbSNP
rs933929999	524	dbSNP
rs1321296865	525	dbSNP
rs748924290	541	dbSNP
rs1376843992	547	dbSNP
rs901108008	550	dbSNP
rs1001886274	553	dbSNP
rs904869335	558	dbSNP
rs528057745	566	dbSNP
rs1390164847	570	dbSNP
rs78977115	572	dbSNP
rs748880781	579	dbSNP
rs1444668196	580	dbSNP
rs1278500962	581	dbSNP
rs1053096715	585	dbSNP
rs1401363949	586	dbSNP
rs1175762589	591	dbSNP
rs982364628	602	dbSNP
rs556809550	605	dbSNP
rs542512204	606	dbSNP
rs573506354	607	dbSNP
rs1251035650	608	dbSNP
rs1432405812	612	dbSNP
rs1418793006	614	dbSNP
rs777305897	623	dbSNP
rs1196430415	624	dbSNP
rs2292811	630	dbSNP
rs1434936036	634	dbSNP
rs942724487	645	dbSNP
rs963517428	646	dbSNP
rs148155847	649	dbSNP
rs1460964447	655	dbSNP
rs1016637873	656	dbSNP
rs912576539	657	dbSNP
rs1238449644	658	dbSNP
rs1005293128	659	dbSNP
rs1301627654	662	dbSNP
rs1320871857	663	dbSNP
rs889437572	672	dbSNP
rs1235288120	677	dbSNP
rs986857103	685	dbSNP
rs951437717	688	dbSNP
rs1026256873	691	dbSNP
rs972362577	694	dbSNP
rs963200706	696	dbSNP
rs1488084740	697	dbSNP
rs1225296616	709	dbSNP
rs575665191	716	dbSNP
rs1454476635	721	dbSNP
rs558761920	729	dbSNP
rs1187521192	732	dbSNP
rs1387887764	733	dbSNP
rs1429812631	743	dbSNP
rs539058910	768	dbSNP
rs1016010769	769	dbSNP
rs1230130835	771	dbSNP
rs566748779	776	dbSNP
rs904985486	778	dbSNP
rs553705233	790	dbSNP
rs768125116	791	dbSNP
rs1397098790	795	dbSNP
rs1450471286	797	dbSNP
rs1408400285	798	dbSNP
rs1441978929	802	dbSNP
rs1303892727	807	dbSNP
rs1360084307	810	dbSNP
rs755581661	817	dbSNP
rs7247146	820	dbSNP
rs1231349563	823	dbSNP
rs1362615509	827	dbSNP
rs567873919	829	dbSNP
rs143812338	830	dbSNP
rs1053856842	833	dbSNP
rs530988193	834	dbSNP
rs1186653409	838	dbSNP
rs149018373	848	dbSNP
rs936898676	852	dbSNP
rs1054044374	853	dbSNP
rs938098102	856	dbSNP
rs778733943	857	dbSNP
rs926749274	858	dbSNP
rs1039862266	862	dbSNP
rs1462323787	865	dbSNP
rs1158803259	868	dbSNP
rs942840932	872	dbSNP
rs551176563	873	dbSNP
rs1378504943	885	dbSNP
rs912672983	886	dbSNP
rs1295091100	890	dbSNP
rs1335326336	891	dbSNP
rs1227575960	892	dbSNP
rs987274069	893	dbSNP
rs1258961654	894	dbSNP
rs949422560	906	dbSNP
rs930004318	910	dbSNP
rs918558890	913	dbSNP
rs919465834	917	dbSNP
rs972008932	923	dbSNP
rs972329241	939	dbSNP
rs528136531	940	dbSNP
rs1177060373	942	dbSNP
rs1354170846	943	dbSNP
rs963229520	944	dbSNP
rs1016126885	950	dbSNP
rs1375034714	955	dbSNP
rs909414728	959	dbSNP
rs1314769949	971	dbSNP
rs983817216	972	dbSNP
rs1385015580	973	dbSNP
rs1313499679	977	dbSNP
rs1375224344	978	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.