ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gaGGUAAAAGGUG-GU--CUCGACGu 5' | | ||| |:: || ||||||| Target 5' ctCAACTTTACGTGCACTGAGCTGCa 3'	261 - 286	155.00	-20.40
2	miRNA 3' gaGGUAAAAGGU-----GGUCUCGACgu 5' :| | ||||| ::|:|||| Target 5' ttTCTTGTTCCAGAGAGGTGGGGCTGga 3'	218 - 245	117.00	-13.60
3	miRNA 3' gaGGUAAAAGGUG-GUCUCGACGu 5' || || |||: | || |||: Target 5' ccCCTGTTCCCATGCTGACCTGTg 3'	177 - 200	113.00	-10.19

Mutant ID	Mutant Position	Mutant Source
COSN19766120	36	COSMIC
COSN28652755	40	COSMIC
COSN30735378	42	COSMIC
COSN8514018	52	COSMIC
COSN2531137	55	COSMIC
COSN28648147	131	COSMIC
COSN30513989	192	COSMIC
COSN26978908	193	COSMIC
COSN23739979	195	COSMIC
COSN30531571	212	COSMIC
COSN24332631	213	COSMIC
COSN19665295	223	COSMIC
COSN18731059	234	COSMIC
COSN19665294	285	COSMIC
COSN28828620	302	COSMIC
COSN30178591	306	COSMIC
COSN19641194	344	COSMIC
COSN19641623	345	COSMIC
COSN18899423	416	COSMIC
COSN18779926	419	COSMIC
COSN7870421	443	COSMIC
COSN32063913	536	COSMIC
COSN32063912	537	COSMIC
COSN9905975	552	COSMIC
COSN32057432	574	COSMIC
COSN32064934	579	COSMIC
rs2428494	63	GWAS
rs3819306	127	GWAS
rs17192932	131	GWAS
rs1055348	312	GWAS
rs1055821	341	GWAS
rs1093	354	GWAS

Mutant ID	Mutant Position	Mutant Source
rs1337747386	4	dbSNP
rs1456440504	15	dbSNP
rs554375828	16	dbSNP
rs757784333	19	dbSNP
rs752043040	23	dbSNP
rs777309025	25	dbSNP
rs757902850	27	dbSNP
rs1334701691	28	dbSNP
rs368900262	29	dbSNP
rs758113479	29	dbSNP
rs752455113	30	dbSNP
rs3819301	31	dbSNP
rs773586936	31	dbSNP
rs151341397	32	dbSNP
rs371453198	32	dbSNP
rs762362662	32	dbSNP
rs760742764	33	dbSNP
rs774618106	33	dbSNP
rs764334129	34	dbSNP
rs768854353	34	dbSNP
rs749582858	35	dbSNP
rs762927419	35	dbSNP
rs146647111	36	dbSNP
rs151341398	36	dbSNP
rs151341399	36	dbSNP
rs200601442	36	dbSNP
rs746944105	37	dbSNP
rs757915336	37	dbSNP
rs777029377	37	dbSNP
rs1416763179	38	dbSNP
rs1491233979	38	dbSNP
rs1491494411	38	dbSNP
rs777319690	38	dbSNP
rs151341404	39	dbSNP
rs370667462	39	dbSNP
rs752136964	39	dbSNP
rs755662330	39	dbSNP
rs200450928	40	dbSNP
rs2523610	40	dbSNP
rs55883829	40	dbSNP
rs1389528276	41	dbSNP
rs752102489	41	dbSNP
rs762480686	41	dbSNP
rs764640734	42	dbSNP
rs778242050	42	dbSNP
rs151341409	43	dbSNP
rs151341411	44	dbSNP
rs539008827	44	dbSNP
rs763154298	44	dbSNP
rs769954870	44	dbSNP
rs775588673	44	dbSNP
rs776355424	44	dbSNP
rs151341413	45	dbSNP
rs746747644	45	dbSNP
rs770746820	45	dbSNP
rs777690969	45	dbSNP
rs386699129	46	dbSNP
rs747681967	46	dbSNP
rs771958279	46	dbSNP
rs778443302	46	dbSNP
rs778465409	46	dbSNP
rs780785232	46	dbSNP
rs281864648	47	dbSNP
rs753528359	47	dbSNP
rs754606020	47	dbSNP
rs781470237	47	dbSNP
rs9279150	47	dbSNP
rs1472476753	48	dbSNP
rs751928955	48	dbSNP
rs753503565	48	dbSNP
rs137854846	49	dbSNP
rs752755390	49	dbSNP
rs763528338	49	dbSNP
rs764718001	49	dbSNP
rs151341415	50	dbSNP
rs765353562	50	dbSNP
rs776922644	50	dbSNP
rs71920763	51	dbSNP
rs760496154	51	dbSNP
rs771073034	51	dbSNP
rs9266106	51	dbSNP
rs13210800	52	dbSNP
rs201315090	52	dbSNP
rs386699128	52	dbSNP
rs1250836335	53	dbSNP
rs1305935653	53	dbSNP
rs1453438027	53	dbSNP
rs386699126	53	dbSNP
rs386699127	53	dbSNP
rs762981586	53	dbSNP
rs879870019	53	dbSNP
rs1410211688	54	dbSNP
rs151341417	54	dbSNP
rs1289174136	55	dbSNP
rs151341416	55	dbSNP
rs151341418	55	dbSNP
rs9279149	55	dbSNP
rs1208411534	56	dbSNP
rs1357656160	56	dbSNP
rs1468794580	56	dbSNP
rs199966337	56	dbSNP
rs386699123	56	dbSNP
rs386699124	56	dbSNP
rs386699125	56	dbSNP
rs1434869758	57	dbSNP
rs201499799	58	dbSNP
rs550237956	58	dbSNP
rs1363412379	59	dbSNP
rs150296664	60	dbSNP
rs2428494	63	dbSNP
rs531022637	64	dbSNP
rs189884234	67	dbSNP
rs1431059870	74	dbSNP
rs185308624	76	dbSNP
rs1318768549	85	dbSNP
rs2428495	85	dbSNP
rs13210793	86	dbSNP
rs71533897	87	dbSNP
rs938882279	89	dbSNP
rs979542815	95	dbSNP
rs561081099	97	dbSNP
rs1369904739	98	dbSNP
rs1197463958	102	dbSNP
rs151341420	106	dbSNP
rs1167839022	107	dbSNP
rs559352471	108	dbSNP
rs1023670956	109	dbSNP
rs927448325	110	dbSNP
rs3819305	116	dbSNP
rs17199279	117	dbSNP
rs1235591093	120	dbSNP
rs1208121793	121	dbSNP
rs151341421	123	dbSNP
rs1256431742	124	dbSNP
rs3819306	127	dbSNP
rs17192932	131	dbSNP
rs151341422	135	dbSNP
rs897672239	138	dbSNP
rs2428496	139	dbSNP
rs1006002378	141	dbSNP
rs572049214	143	dbSNP
rs1454624545	144	dbSNP
rs1295545383	145	dbSNP
rs1468333265	146	dbSNP
rs1378006895	147	dbSNP
rs111301312	152	dbSNP
rs1464763874	156	dbSNP
rs1170445918	165	dbSNP
rs1017439212	166	dbSNP
rs1209899339	167	dbSNP
rs1050118239	173	dbSNP
rs931658038	178	dbSNP
rs903773600	180	dbSNP
rs879226458	181	dbSNP
rs878896939	182	dbSNP
rs878931748	183	dbSNP
rs112353744	184	dbSNP
rs187858816	187	dbSNP
rs1188129693	188	dbSNP
rs202110486	193	dbSNP
rs1432333791	195	dbSNP
rs1176582628	200	dbSNP
rs1430205680	204	dbSNP
rs1471115178	204	dbSNP
rs571120465	207	dbSNP
rs1053726	213	dbSNP
rs1291107308	214	dbSNP
rs1366145540	216	dbSNP
rs879003426	220	dbSNP
rs1170897250	222	dbSNP
rs111706358	223	dbSNP
rs1325238968	232	dbSNP
rs112860077	233	dbSNP
rs1054111	234	dbSNP
rs1217338588	236	dbSNP
rs1244887744	238	dbSNP
rs570197742	239	dbSNP
rs1191548996	245	dbSNP
rs916551364	249	dbSNP
rs151341423	256	dbSNP
rs1054338	257	dbSNP
rs1471493276	262	dbSNP
rs990815437	275	dbSNP
rs1411549727	276	dbSNP
rs748390639	285	dbSNP
rs1161440244	286	dbSNP
rs1056240556	293	dbSNP
rs71563314	299	dbSNP
rs1002795639	300	dbSNP
rs1055149	302	dbSNP
rs1014787514	303	dbSNP
rs1408419640	305	dbSNP
rs1287267095	306	dbSNP
rs1055348	312	dbSNP
rs112978603	322	dbSNP
rs1321034513	330	dbSNP
rs140769830	335	dbSNP
rs57138251	335	dbSNP
rs1293868475	337	dbSNP
rs1049645	338	dbSNP
rs1181218148	339	dbSNP
rs770267712	340	dbSNP
rs1055821	341	dbSNP
rs117007172	343	dbSNP
rs1055849	344	dbSNP
rs1055890	345	dbSNP
rs386699122	345	dbSNP
rs879124740	345	dbSNP
rs1398553105	346	dbSNP
rs386699121	346	dbSNP
rs879609258	346	dbSNP
rs1298644089	350	dbSNP
rs1093	354	dbSNP
rs1017661886	361	dbSNP
rs1443996320	362	dbSNP
rs1282078601	363	dbSNP
rs281864649	363	dbSNP
rs759885434	364	dbSNP
rs1166614068	366	dbSNP
rs1476346543	369	dbSNP
rs572137225	370	dbSNP
rs1181562901	371	dbSNP
rs1471612632	372	dbSNP
rs902677532	373	dbSNP
rs1043923656	376	dbSNP
rs1056429	377	dbSNP
rs2769	378	dbSNP
rs991322166	385	dbSNP
rs1387594377	389	dbSNP
rs1264126693	393	dbSNP
rs1215930005	394	dbSNP
rs763249433	396	dbSNP
rs927982665	398	dbSNP
rs2768	404	dbSNP
rs361531	415	dbSNP
rs114910620	416	dbSNP
rs1307062218	419	dbSNP
rs1362940879	421	dbSNP
rs139093151	423	dbSNP
rs11546717	426	dbSNP
rs1207820266	429	dbSNP
rs537380578	431	dbSNP
rs1341132017	435	dbSNP
rs62396266	437	dbSNP
rs1276742292	439	dbSNP
rs1204380468	445	dbSNP
rs1327735783	447	dbSNP
rs1461712630	448	dbSNP
rs951978894	451	dbSNP
rs2770	453	dbSNP
rs1056978	454	dbSNP
rs1171245821	455	dbSNP
rs3180225	457	dbSNP
rs1470059126	463	dbSNP
rs1338115105	468	dbSNP
rs1385735014	470	dbSNP
rs1433719413	474	dbSNP
rs1018594785	478	dbSNP
rs374112227	480	dbSNP
rs565793170	480	dbSNP
rs1057151	484	dbSNP
rs10947205	485	dbSNP
rs3177766	490	dbSNP
rs3177764	491	dbSNP
rs3177763	494	dbSNP
rs1224291401	497	dbSNP
rs1433175024	504	dbSNP
rs1057387	507	dbSNP
rs1057412	508	dbSNP
rs1469156421	515	dbSNP
rs191345591	529	dbSNP
rs1055214796	533	dbSNP
rs879240746	536	dbSNP
rs1260876937	537	dbSNP
rs3177757	539	dbSNP
rs1268698229	552	dbSNP
rs186928221	560	dbSNP
rs574360302	563	dbSNP
rs878941341	566	dbSNP
rs1456227581	567	dbSNP
rs542419099	569	dbSNP
rs1049268	574	dbSNP
rs1058026	575	dbSNP
rs1058067	579	dbSNP
rs879032600	579	dbSNP
rs985114885	580	dbSNP
rs1345832296	585	dbSNP
rs1318268468	588	dbSNP
rs1406572751	594	dbSNP
rs1285372580	597	dbSNP
rs1348920732	602	dbSNP
rs3177747	603	dbSNP
rs577699292	608	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.