>YARS|NM_003680|3'UTR
   1 CCAGCCCAGCATCTTCCCCCCTTCTTCCACCACTGAGTCATCTGCTGTCTCTTCAGTCTGCTCCATCCATCACCCATTTA
  81 CCCATCTCTCAGGACACGGAAGCAGCGGGTTTGGACTCTTTATTCGGTGCAGAACTCGGCAAGGGGCAGCTTACCCTCCC
 161 CAGAACCCAGGATCATCCTGTCTGGCTGCAGTGAGAGACCAACCCCTAACAAGGGCTGGGCCACAGCAGGGAGTCCAGCC
 241 CTACCTTCTTCCCTTGGCAGCTGGAGAAATCTGGTTTCAATATAACTCATTTAAAAATTTATGCCACAGTCCTTATAATT
 321 GGAAAAATACTGGTGCCCAGGTTTTCTTGGAGTTATCCAAGCAGCTGCGCCCCTAGCTGGGATCTGGTACCTGGACTAGG
 401 CTAATTACAGCTTCTCCCCAACAGGAAACTGTGGGATTTGAAAAGGAAAGGGAAGGGAAAACAGAGAACCTAGTGGTCTA
 481 CCAAGTGGTTGGCAACTTTCCCAATGTCTGCTTACTCTGAGGCTTGGCACTGGGGGCCAGGGCCTGCCCCAGGGCTCCTG
 561 GAATTTCCCTTGATCCAGCTAGGCTGGGACACTCCCTAAATCAGCTGCGTGTTGTTAGCATCAGGCAGAATGAATGGCAG
 641 AGAGTGATTCTGTCTTCATAGAGGGTGGGGTACTTCTCCATAAGGCATCTCAGTCAAATCCCCATCACTGTCATAAATTC
 721 AAATAAAATGTCTGAACAAGGG

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uuACUGG--GAUGGAUAAGUCAGuc 5' | |:| ||::|| ||||||| Target 5' agTCATCTGCTGTCTCTTCAGTCtg 3'	36 - 60	147.00	-16.20
2	miRNA 3' uuacUG-GGAUGGA-----UA-AGUCAGUc 5' || :|| | | || ||||||| Target 5' gggtACTTCTCCATAAGGCATCTCAGTCAa 3'	668 - 697	143.00	-9.30
3	miRNA 3' uuaCUGGGAUGGAUAAGUCA-GUc 5' |||:|| :|||||:|| || Target 5' ttgGACTCT--TTATTCGGTGCAg 3'	111 - 132	124.00	-19.60

Mutant ID	Mutant Position	Mutant Source
297147	21	ClinVar
1200883	106	ClinVar
297146	138	ClinVar
297145	284	ClinVar
297144	293	ClinVar
297143	309	ClinVar
297142	369	ClinVar
297141	386	ClinVar
297140	399	ClinVar
297139	439	ClinVar
297138	452	ClinVar
297137	539	ClinVar
297136	599	ClinVar
297135	680	ClinVar
297134	681	ClinVar
297133	724	ClinVar
COSN30190822	6	COSMIC
COSN19735355	14	COSMIC
COSN30616847	16	COSMIC
COSN30483276	18	COSMIC
COSN2530893	28	COSMIC
COSN30154347	54	COSMIC
COSN30154337	58	COSMIC
COSN30509607	64	COSMIC
COSN26599274	197	COSMIC
COSN31513900	299	COSMIC
COSN31544438	357	COSMIC
COSN25221884	555	COSMIC
COSN32008113	605	COSMIC
COSN5371074	648	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs771314633	4	dbSNP
rs1452443769	5	dbSNP
rs1188754782	14	dbSNP
rs1249827285	15	dbSNP
rs749697240	16	dbSNP
rs777955605	18	dbSNP
rs781560790	20	dbSNP
rs149812692	21	dbSNP
rs758807329	22	dbSNP
rs1313051365	24	dbSNP
rs1245862756	25	dbSNP
rs1300619730	27	dbSNP
rs1448765809	32	dbSNP
rs750871661	33	dbSNP
rs751669673	34	dbSNP
rs1205683945	35	dbSNP
rs765250908	47	dbSNP
rs1208006443	55	dbSNP
rs1268779294	58	dbSNP
rs1276902575	59	dbSNP
rs933078773	69	dbSNP
rs1364704912	71	dbSNP
rs924396529	77	dbSNP
rs1022517973	81	dbSNP
rs1439035416	88	dbSNP
rs1220436664	97	dbSNP
rs538986203	98	dbSNP
rs112037794	106	dbSNP
rs760241532	107	dbSNP
rs1204796554	108	dbSNP
rs959497468	110	dbSNP
rs1428584051	111	dbSNP
rs1387927529	114	dbSNP
rs1034895128	116	dbSNP
rs1478202329	117	dbSNP
rs1245920565	119	dbSNP
rs1196616499	122	dbSNP
rs190556254	125	dbSNP
rs1351104166	126	dbSNP
rs1282568422	137	dbSNP
rs41265857	138	dbSNP
rs1271077097	139	dbSNP
rs886402993	147	dbSNP
rs1047728933	148	dbSNP
rs981517291	160	dbSNP
rs995303205	169	dbSNP
rs538069830	193	dbSNP
rs1346099912	199	dbSNP
rs1274652735	202	dbSNP
rs772114341	204	dbSNP
rs35504526	207	dbSNP
rs1039551397	210	dbSNP
rs567446820	213	dbSNP
rs1309235677	218	dbSNP
rs1408670624	220	dbSNP
rs943536356	221	dbSNP
rs912098353	226	dbSNP
rs1025756048	228	dbSNP
rs1052014226	237	dbSNP
rs1414716178	242	dbSNP
rs995493162	244	dbSNP
rs934900115	245	dbSNP
rs1053448	248	dbSNP
rs1170573409	252	dbSNP
rs544493865	260	dbSNP
rs575566299	261	dbSNP
rs1007891148	266	dbSNP
rs1428947029	273	dbSNP
rs1197906508	280	dbSNP
rs968176690	282	dbSNP
rs150690731	284	dbSNP
rs1212446414	288	dbSNP
rs1443191058	289	dbSNP
rs1052024512	291	dbSNP
rs16866009	293	dbSNP
rs180881898	297	dbSNP
rs1192535032	301	dbSNP
rs370883906	308	dbSNP
rs372462149	309	dbSNP
rs1289466316	320	dbSNP
rs1228328357	325	dbSNP
rs1286606510	331	dbSNP
rs1041477747	334	dbSNP
rs778815402	335	dbSNP
rs1034821441	337	dbSNP
rs1003382031	342	dbSNP
rs1304105817	346	dbSNP
rs950649114	349	dbSNP
rs1026250916	368	dbSNP
rs879511403	369	dbSNP
rs1329624042	382	dbSNP
rs144131403	386	dbSNP
rs1412439810	387	dbSNP
rs1458605425	387	dbSNP
rs1039109057	391	dbSNP
rs1158280425	396	dbSNP
rs1221649828	397	dbSNP
rs1008095662	398	dbSNP
rs1057515511	399	dbSNP
rs890588915	399	dbSNP
rs937599919	408	dbSNP
rs189698373	418	dbSNP
rs1429393583	421	dbSNP
rs1237102040	425	dbSNP
rs532734575	426	dbSNP
rs1466480263	432	dbSNP
rs1269198765	434	dbSNP
rs1324391190	436	dbSNP
rs184249741	439	dbSNP
rs1315487563	441	dbSNP
rs981606818	446	dbSNP
rs142185532	452	dbSNP
rs1312373353	462	dbSNP
rs1405836726	464	dbSNP
rs1436871132	466	dbSNP
rs934868758	479	dbSNP
rs748331867	489	dbSNP
rs1319617578	492	dbSNP
rs765324724	497	dbSNP
rs1351541237	499	dbSNP
rs1165463409	501	dbSNP
rs1424409705	506	dbSNP
rs1413176664	515	dbSNP
rs1471271189	522	dbSNP
rs758419455	529	dbSNP
rs1181940401	538	dbSNP
rs550239801	539	dbSNP
rs1167478575	548	dbSNP
rs1214600395	552	dbSNP
rs531834594	566	dbSNP
rs1043733434	572	dbSNP
rs561525459	574	dbSNP
rs1188294908	575	dbSNP
rs1485136737	580	dbSNP
rs542816112	599	dbSNP
rs962700279	607	dbSNP
rs1320548817	608	dbSNP
rs1288984333	609	dbSNP
rs1230037058	616	dbSNP
rs1365432361	618	dbSNP
rs1296497267	624	dbSNP
rs914933026	625	dbSNP
rs1243862713	629	dbSNP
rs1380296057	637	dbSNP
rs1189484027	640	dbSNP
rs1461010733	644	dbSNP
rs12145	650	dbSNP
rs1018698842	659	dbSNP
rs1053482	661	dbSNP
rs1457377559	665	dbSNP
rs1006925839	667	dbSNP
rs1161726404	672	dbSNP
rs536402126	680	dbSNP
rs78319035	681	dbSNP
rs1188231936	682	dbSNP
rs981930420	685	dbSNP
rs1213850380	688	dbSNP
rs573873811	700	dbSNP
rs1267427869	707	dbSNP
rs1206673241	713	dbSNP
rs1342324573	717	dbSNP
rs1273387259	720	dbSNP
rs1057515549	724	dbSNP
rs950577466	724	dbSNP
rs1026216397	729	dbSNP
rs1313702790	731	dbSNP
rs1301750102	739	dbSNP
rs1470091199	743	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.