ID	Duplex structure	Position	Score	MFE
1	miRNA 3' agacuaguccguuucAACGUCc 5' |||||| Target 5' tccattcctctgcacTTGCAGa 3'	94 - 115	120.00	-7.60
2	miRNA 3' agaCUAGUCCGUUU-----CAACGUCc 5' ||| :||| :: | ||||| Target 5' ggaGATGGGGCTGGTTCCTGCTGCAGt 3'	562 - 588	119.00	-12.40
3	miRNA 3' agacUAG--UCCGUUU-CAACGUcc 5' ||| :|||||| ||| :| Target 5' cactATCCTGGGCAAATGTTTTAcc 3'	312 - 336	100.00	-7.70

Mutant ID	Mutant Position	Mutant Source
892417	28	ClinVar
325522	106	ClinVar
892418	136	ClinVar
325523	256	ClinVar
325524	309	ClinVar
889035	400	ClinVar
325525	463	ClinVar
325526	523	ClinVar
889036	653	ClinVar
COSM7636476	1	COSMIC
COSN30462579	13	COSMIC
COSN506156	23	COSMIC
COSN28712538	142	COSMIC
COSN26434208	169	COSMIC
COSN30707828	224	COSMIC
COSN27624283	283	COSMIC
COSN6660929	309	COSMIC
COSN31597411	626	COSMIC
rs4227	309	GWAS
rs545206972	463	GWAS

Mutant ID	Mutant Position	Mutant Source
rs987746341	1	dbSNP
rs1191976207	4	dbSNP
rs1372905442	5	dbSNP
rs1282453826	7	dbSNP
rs755001933	10	dbSNP
rs753997450	11	dbSNP
rs757375576	12	dbSNP
rs1424961136	23	dbSNP
rs370530785	24	dbSNP
rs374857107	28	dbSNP
rs758537958	29	dbSNP
rs1387604862	32	dbSNP
rs780535701	33	dbSNP
rs747344078	39	dbSNP
rs769173532	41	dbSNP
rs1278528496	42	dbSNP
rs200572297	45	dbSNP
rs371831033	48	dbSNP
rs747661270	50	dbSNP
rs1488501976	51	dbSNP
rs1204231063	52	dbSNP
rs368683775	55	dbSNP
rs951520479	57	dbSNP
rs1306539967	60	dbSNP
rs769288328	76	dbSNP
rs1196318791	79	dbSNP
rs772580910	80	dbSNP
rs1264289987	85	dbSNP
rs1471140088	87	dbSNP
rs762730294	88	dbSNP
rs1163259792	93	dbSNP
rs1177839815	99	dbSNP
rs937440050	102	dbSNP
rs765911922	103	dbSNP
rs537710358	104	dbSNP
rs190574792	106	dbSNP
rs781336281	109	dbSNP
rs1287864307	112	dbSNP
rs757428529	117	dbSNP
rs564782224	121	dbSNP
rs750465889	126	dbSNP
rs758686598	130	dbSNP
rs1394260231	131	dbSNP
rs780090441	136	dbSNP
rs747405299	143	dbSNP
rs1237693854	146	dbSNP
rs755266911	149	dbSNP
rs781672491	154	dbSNP
rs1216841384	159	dbSNP
rs532280586	165	dbSNP
rs1483116369	166	dbSNP
rs1180683928	170	dbSNP
rs1258869295	171	dbSNP
rs748710016	173	dbSNP
rs1326936124	175	dbSNP
rs1178114059	181	dbSNP
rs1458137818	184	dbSNP
rs1173585188	187	dbSNP
rs949887102	188	dbSNP
rs1421785805	189	dbSNP
rs1162162044	190	dbSNP
rs1159441920	193	dbSNP
rs1047002917	195	dbSNP
rs1380314524	196	dbSNP
rs373333441	203	dbSNP
rs1330804679	211	dbSNP
rs1464341442	213	dbSNP
rs74965548	215	dbSNP
rs376449010	217	dbSNP
rs1390702995	219	dbSNP
rs1306496816	222	dbSNP
rs1038460637	224	dbSNP
rs559401007	228	dbSNP
rs1279146605	231	dbSNP
rs530076129	235	dbSNP
rs935173887	236	dbSNP
rs899467428	241	dbSNP
rs1258077690	247	dbSNP
rs1483876844	250	dbSNP
rs878936939	256	dbSNP
rs758284391	259	dbSNP
rs1238642576	266	dbSNP
rs770642873	268	dbSNP
rs942581931	273	dbSNP
rs1179554718	274	dbSNP
rs1424715177	276	dbSNP
rs1431586690	277	dbSNP
rs1177978962	278	dbSNP
rs368564787	278	dbSNP
rs1359488796	279	dbSNP
rs548294559	280	dbSNP
rs1050811090	281	dbSNP
rs1039734869	288	dbSNP
rs1401918751	289	dbSNP
rs1227722188	296	dbSNP
rs1301982580	298	dbSNP
rs890667024	304	dbSNP
rs1220546085	307	dbSNP
rs4227	309	dbSNP
rs746850584	318	dbSNP
rs1218615111	319	dbSNP
rs1256697386	322	dbSNP
rs1459195254	323	dbSNP
rs1176599289	331	dbSNP
rs968053961	336	dbSNP
rs1188543055	338	dbSNP
rs1247691412	339	dbSNP
rs1000424656	340	dbSNP
rs768271659	341	dbSNP
rs1487647937	343	dbSNP
rs1214498491	344	dbSNP
rs767366072	345	dbSNP
rs1425674336	347	dbSNP
rs1477367388	349	dbSNP
rs1168531467	350	dbSNP
rs1425695127	351	dbSNP
rs1466025978	352	dbSNP
rs1322333208	353	dbSNP
rs1347569923	356	dbSNP
rs1438217500	361	dbSNP
rs1033456781	365	dbSNP
rs1293730940	379	dbSNP
rs1298133108	379	dbSNP
rs775414004	383	dbSNP
rs958680293	384	dbSNP
rs1297115928	390	dbSNP
rs374315569	390	dbSNP
rs114118916	391	dbSNP
rs1219730140	392	dbSNP
rs1289297756	400	dbSNP
rs1003060823	402	dbSNP
rs1224135180	403	dbSNP
rs1247505623	406	dbSNP
rs1488048484	408	dbSNP
rs1191254642	410	dbSNP
rs1409333737	411	dbSNP
rs1265003037	412	dbSNP
rs1465451448	415	dbSNP
rs1478123985	416	dbSNP
rs1162551977	422	dbSNP
rs1323909948	423	dbSNP
rs1172246797	424	dbSNP
rs1411057295	425	dbSNP
rs1478292388	430	dbSNP
rs552782991	432	dbSNP
rs1165120908	436	dbSNP
rs1359497255	439	dbSNP
rs1397683772	440	dbSNP
rs1200416079	441	dbSNP
rs1482534668	442	dbSNP
rs750566373	444	dbSNP
rs1253500470	445	dbSNP
rs971422807	446	dbSNP
rs1318231739	448	dbSNP
rs34052966	453	dbSNP
rs571090952	456	dbSNP
rs959016999	457	dbSNP
rs1395437559	460	dbSNP
rs1284116581	462	dbSNP
rs545206972	463	dbSNP
rs908346568	464	dbSNP
rs1272040378	466	dbSNP
rs1337975287	467	dbSNP
rs941287199	472	dbSNP
rs1290612672	474	dbSNP
rs1214527916	475	dbSNP
rs1353289707	476	dbSNP
rs1214677671	479	dbSNP
rs1287943843	487	dbSNP
rs751721516	488	dbSNP
rs747826630	489	dbSNP
rs1235658992	501	dbSNP
rs968109714	503	dbSNP
rs1422653622	508	dbSNP
rs1473696851	509	dbSNP
rs1181948214	510	dbSNP
rs1379098306	514	dbSNP
rs932303177	518	dbSNP
rs1050864392	523	dbSNP
rs543253078	523	dbSNP
rs886053483	523	dbSNP
rs1009280355	524	dbSNP
rs1042292152	531	dbSNP
rs1333850527	532	dbSNP
rs1339241031	535	dbSNP
rs1450755496	536	dbSNP
rs1315862185	538	dbSNP
rs1376209986	539	dbSNP
rs1238306379	542	dbSNP
rs1311721784	544	dbSNP
rs1354233737	551	dbSNP
rs556327306	552	dbSNP
rs1030708579	553	dbSNP
rs553603161	557	dbSNP
rs1375917544	558	dbSNP
rs958735713	559	dbSNP
rs1488434408	560	dbSNP
rs1012880356	561	dbSNP
rs1237088784	566	dbSNP
rs1437265893	567	dbSNP
rs1024222099	568	dbSNP
rs1180199216	570	dbSNP
rs568664507	572	dbSNP
rs769224800	576	dbSNP
rs1172384808	578	dbSNP
rs1400467714	579	dbSNP
rs182362947	582	dbSNP
rs756688946	583	dbSNP
rs1337937934	585	dbSNP
rs772609484	587	dbSNP
rs982856614	589	dbSNP
rs1399721436	592	dbSNP
rs1236789618	598	dbSNP
rs908398461	601	dbSNP
rs1444649016	603	dbSNP
rs1333371061	608	dbSNP
rs778097197	611	dbSNP
rs1232741737	613	dbSNP
rs1208809251	614	dbSNP
rs1272887078	618	dbSNP
rs1346820178	619	dbSNP
rs1224111496	625	dbSNP
rs536206425	628	dbSNP
rs770282612	631	dbSNP
rs778725487	636	dbSNP
rs3211115	638	dbSNP
rs1352867273	640	dbSNP
rs1365156537	643	dbSNP
rs745574848	644	dbSNP
rs771848986	645	dbSNP
rs1196271510	646	dbSNP
rs554187980	653	dbSNP
rs1479200880	662	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.