ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ugAAAGGGAAGUCUCGGACCGa 5' | |||| || ||||||| Target 5' ctTCTCCCCTCCCCGCCTGGCc 3'	613 - 634	160.00	-19.20
2	miRNA 3' ugaaAGGGA-AGUCU-CGGACCga 5' ||| | :||| |||||| Target 5' ggggTCCGTGGTAGAGGCCTGGgg 3'	759 - 782	134.00	-18.10
3	miRNA 3' ugaaagggaaGUCUCGGACCGa 5' ||||||||| | Target 5' cggcagaaaaCAGAGCCTGCCt 3'	202 - 223	128.00	-17.60

Mutant ID	Mutant Position	Mutant Source
COSN29109745	85	COSMIC
COSN31813219	85	COSMIC
COSN31589948	157	COSMIC
COSN229519	247	COSMIC
COSN20090819	310	COSMIC
COSN28198058	330	COSMIC
COSN31485958	559	COSMIC
COSN31483489	608	COSMIC
COSN28778305	611	COSMIC
COSN20578558	624	COSMIC
COSN31512763	736	COSMIC
COSN23700140	765	COSMIC
COSN31591670	784	COSMIC
COSN1951879	812	COSMIC
COSN16996585	950	COSMIC
COSN16457810	960	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs755520960	3	dbSNP
rs777323151	4	dbSNP
rs755683639	8	dbSNP
rs150672207	9	dbSNP
rs1476888905	10	dbSNP
rs955158633	13	dbSNP
rs998623123	14	dbSNP
rs758942510	18	dbSNP
rs1018163872	20	dbSNP
rs11558285	21	dbSNP
rs973423438	24	dbSNP
rs747331182	31	dbSNP
rs1046959	32	dbSNP
rs1420013816	34	dbSNP
rs1046960	37	dbSNP
rs768955567	38	dbSNP
rs543148563	44	dbSNP
rs1194061636	46	dbSNP
rs1432524177	58	dbSNP
rs1476996064	58	dbSNP
rs1175476079	60	dbSNP
rs1330861833	60	dbSNP
rs1375815358	60	dbSNP
rs1379960466	60	dbSNP
rs1398987225	60	dbSNP
rs1465654023	60	dbSNP
rs148259784	60	dbSNP
rs1491497511	60	dbSNP
rs59949761	60	dbSNP
rs1491108900	61	dbSNP
rs950713961	62	dbSNP
rs952623392	62	dbSNP
rs983888106	64	dbSNP
rs908439766	70	dbSNP
rs908546849	73	dbSNP
rs72205047	75	dbSNP
rs1375426622	82	dbSNP
rs1199338430	84	dbSNP
rs200144707	84	dbSNP
rs992771986	86	dbSNP
rs1045027271	92	dbSNP
rs1308978630	94	dbSNP
rs1431991521	98	dbSNP
rs915714187	101	dbSNP
rs948538869	108	dbSNP
rs1293448934	109	dbSNP
rs976904780	115	dbSNP
rs1395490531	116	dbSNP
rs1400165904	123	dbSNP
rs1356963368	126	dbSNP
rs922768469	146	dbSNP
rs1379689075	151	dbSNP
rs757257202	154	dbSNP
rs1269312818	166	dbSNP
rs1042827610	186	dbSNP
rs1156588794	196	dbSNP
rs1471052307	200	dbSNP
rs1363976665	204	dbSNP
rs1186405974	213	dbSNP
rs1421480346	218	dbSNP
rs904306382	231	dbSNP
rs934330034	234	dbSNP
rs879342730	243	dbSNP
rs139857596	247	dbSNP
rs890144309	249	dbSNP
rs915470956	250	dbSNP
rs1216155430	252	dbSNP
rs1278058491	274	dbSNP
rs1485340582	283	dbSNP
rs1214845773	290	dbSNP
rs1009532991	291	dbSNP
rs1259715781	292	dbSNP
rs1280295446	293	dbSNP
rs1244352313	297	dbSNP
rs1017818530	309	dbSNP
rs550405002	310	dbSNP
rs995001871	314	dbSNP
rs1373945015	315	dbSNP
rs1027683800	320	dbSNP
rs950829557	321	dbSNP
rs983576236	322	dbSNP
rs1415528645	330	dbSNP
rs1476581546	334	dbSNP
rs774090925	335	dbSNP
rs761331446	336	dbSNP
rs369353752	341	dbSNP
rs1035403319	350	dbSNP
rs149702464	351	dbSNP
rs993115795	354	dbSNP
rs1456056960	356	dbSNP
rs999003249	360	dbSNP
rs915871240	361	dbSNP
rs895631699	362	dbSNP
rs948526292	363	dbSNP
rs773649559	369	dbSNP
rs1376752270	373	dbSNP
rs772325544	378	dbSNP
rs1012729896	381	dbSNP
rs1285380307	387	dbSNP
rs1209689246	395	dbSNP
rs925672417	396	dbSNP
rs1354471822	414	dbSNP
rs1022816116	420	dbSNP
rs952701240	421	dbSNP
rs1005467432	422	dbSNP
rs1276160524	425	dbSNP
rs373374707	429	dbSNP
rs1016010132	434	dbSNP
rs1327019279	440	dbSNP
rs1322609436	441	dbSNP
rs961436513	451	dbSNP
rs1396162830	461	dbSNP
rs780391521	468	dbSNP
rs1346986065	479	dbSNP
rs1464591490	479	dbSNP
rs922714638	483	dbSNP
rs954148334	484	dbSNP
rs1243241328	487	dbSNP
rs1052886616	488	dbSNP
rs1198538076	489	dbSNP
rs761264276	498	dbSNP
rs1467440761	500	dbSNP
rs1255045442	507	dbSNP
rs890082734	515	dbSNP
rs944390006	526	dbSNP
rs1270964006	527	dbSNP
rs746058048	539	dbSNP
rs1039257579	551	dbSNP
rs1339965605	555	dbSNP
rs900793450	587	dbSNP
rs946919711	589	dbSNP
rs1262659738	597	dbSNP
rs1042679330	598	dbSNP
rs14321	604	dbSNP
rs1192132925	615	dbSNP
rs1027755767	618	dbSNP
rs1330276119	621	dbSNP
rs530002580	627	dbSNP
rs1455037262	628	dbSNP
rs1176116985	632	dbSNP
rs886622625	633	dbSNP
rs1452898110	635	dbSNP
rs1397253651	637	dbSNP
rs1194725614	638	dbSNP
rs1056834947	651	dbSNP
rs1231438485	656	dbSNP
rs1206651557	663	dbSNP
rs548407997	672	dbSNP
rs1277751323	674	dbSNP
rs1232013637	680	dbSNP
rs1350039287	683	dbSNP
rs1385204141	688	dbSNP
rs1303873428	699	dbSNP
rs1239208106	703	dbSNP
rs1436845738	711	dbSNP
rs1323566139	712	dbSNP
rs1280959280	713	dbSNP
rs1035115881	717	dbSNP
rs768849621	735	dbSNP
rs1350045578	740	dbSNP
rs1370016692	760	dbSNP
rs990871329	763	dbSNP
rs1022873555	765	dbSNP
rs1377836204	766	dbSNP
rs1439491434	767	dbSNP
rs777063502	770	dbSNP
rs1063131	773	dbSNP
rs969985168	774	dbSNP
rs1179177806	780	dbSNP
rs978691283	783	dbSNP
rs754283272	785	dbSNP
rs1236966534	786	dbSNP
rs1046978	790	dbSNP
rs1208908791	794	dbSNP
rs1044210505	795	dbSNP
rs770327830	798	dbSNP
rs1215564889	808	dbSNP
rs773759508	811	dbSNP
rs1308620851	815	dbSNP
rs1223980306	817	dbSNP
rs1290865553	818	dbSNP
rs1378075897	818	dbSNP
rs1416136946	822	dbSNP
rs955906210	824	dbSNP
rs1314050411	825	dbSNP
rs1353045978	829	dbSNP
rs1343819295	831	dbSNP
rs569749749	833	dbSNP
rs1015542805	836	dbSNP
rs1212063350	843	dbSNP
rs530686755	851	dbSNP
rs897131227	853	dbSNP
rs1192870358	854	dbSNP
rs1272903691	857	dbSNP
rs1262265578	859	dbSNP
rs1216280929	862	dbSNP
rs998119298	864	dbSNP
rs1046980	870	dbSNP
rs1029632803	878	dbSNP
rs954095940	879	dbSNP
rs1334927077	880	dbSNP
rs1294384029	885	dbSNP
rs985512558	893	dbSNP
rs1046983	896	dbSNP
rs550016051	901	dbSNP
rs1360576448	903	dbSNP
rs571373906	915	dbSNP
rs538670762	916	dbSNP
rs1195952428	931	dbSNP
rs1022883658	933	dbSNP
rs968273804	934	dbSNP
rs1249024068	949	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.