>GABRB1|NM_000812|3'UTR
   1 GGTCTGTTCTAATGGTTCCATTTAGACTACTTTCCTCTTCTATTGTTTTTTAACCTTACAGGTCCCCAACAGCGATACTG
  81 CTGTTTCTCGAGGTAAGAGATTCAGCCATCCAATTGGTTTTAGGTCTTGCATATCAGTTTTATTACTGCACCATGTTTAC
 161 TTCAAAAAGACAAAACAAAAAAAAAATTATTTTTCCAGTCTACCGTGGTCCAGGTTATCAGCTCTTTAAGAGCTCTATTA
 241 ATTGCCATGTTTACAAACAAACACAAAGAGAGAAGTTAGACAGGTAGATCTTTAGCAGTCTTTTCTAGTTTCCCTGGATT
 321 TCACTGATTTATTTTTTAGGGAAAATGAAAAGAGGACCTTGCTGTCCGCCTGCACTGCTTCCTGGTAAACTATAACAAAC
 401 TTATGCTGCCAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gaguggaCUCGAG-GGCA--CGGAc 5' || ||: |:|| |||| Target 5' aaaagagGACCTTGCTGTCCGCCTg 3'	348 - 372	99.00	-17.40
2	miRNA 3' gaguggacUC-GAGGGCACggac 5' || || ||||| Target 5' atttttccAGTCTACCGTGgtcc 3'	189 - 211	94.00	-10.60
3	miRNA 3' gaGUGGACUC---GAGGGCACGGAc 5' || | | | || |:|| :|| Target 5' ccCAACAGCGATACTGCTGTTTCTc 3'	65 - 89	92.00	-10.50

Mutant ID	Mutant Position	Mutant Source
COSN30188073	3	COSMIC
COSN31600213	5	COSMIC
COSN29284499	7	COSMIC
COSN31501200	14	COSMIC
COSN30183540	19	COSMIC
COSN30514083	25	COSMIC
COSN30152781	26	COSMIC
COSN30102322	38	COSMIC
COSN30450894	41	COSMIC
COSN18729136	45	COSMIC
COSN30146734	67	COSMIC
COSN14113683	69	COSMIC
COSN26976991	74	COSMIC
COSN18718284	75	COSMIC
COSN30186276	79	COSMIC
COSN31612422	79	COSMIC
COSN1297839	85	COSMIC
COSN24383763	90	COSMIC
COSN19709404	91	COSMIC
COSN508369	97	COSMIC
COSN31501623	136	COSMIC
COSN28868144	187	COSMIC
COSN32064928	205	COSMIC
COSN20100028	305	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1471788158	1	dbSNP
rs776456940	2	dbSNP
rs1455729676	5	dbSNP
rs1339197576	7	dbSNP
rs745503672	8	dbSNP
rs1447756614	10	dbSNP
rs1282852852	12	dbSNP
rs769397165	14	dbSNP
rs774995285	16	dbSNP
rs373376360	21	dbSNP
rs769161799	23	dbSNP
rs1205033007	30	dbSNP
rs775033184	39	dbSNP
rs762453818	43	dbSNP
rs1185130440	44	dbSNP
rs376352619	45	dbSNP
rs1465023387	46	dbSNP
rs750877808	52	dbSNP
rs1464885482	55	dbSNP
rs1272951018	60	dbSNP
rs1329092137	63	dbSNP
rs544199293	69	dbSNP
rs1359099026	70	dbSNP
rs899694880	71	dbSNP
rs1036432184	75	dbSNP
rs553175666	79	dbSNP
rs997999417	88	dbSNP
rs1299846916	91	dbSNP
rs1436097864	96	dbSNP
rs762537059	98	dbSNP
rs1393974496	105	dbSNP
rs1324994702	109	dbSNP
rs1403369540	110	dbSNP
rs897903968	111	dbSNP
rs577415550	114	dbSNP
rs1474827414	130	dbSNP
rs1004502912	132	dbSNP
rs1189863306	144	dbSNP
rs1220012571	150	dbSNP
rs763675411	152	dbSNP
rs963346310	156	dbSNP
rs1453650700	159	dbSNP
rs570487863	164	dbSNP
rs1316975291	170	dbSNP
rs1197107518	172	dbSNP
rs1337947443	174	dbSNP
rs111359296	177	dbSNP
rs1276905715	177	dbSNP
rs1370594954	177	dbSNP
rs569240613	177	dbSNP
rs1281107055	180	dbSNP
rs1301489235	181	dbSNP
rs889382217	182	dbSNP
rs1332592106	185	dbSNP
rs1209948442	188	dbSNP
rs1409304057	189	dbSNP
rs192098838	205	dbSNP
rs1021867588	208	dbSNP
rs1034384807	215	dbSNP
rs1394139051	219	dbSNP
rs968402155	231	dbSNP
rs575157459	233	dbSNP
rs1194535350	238	dbSNP
rs1247664284	242	dbSNP
rs1181489737	244	dbSNP
rs993031570	263	dbSNP
rs1206009073	265	dbSNP
rs1476938820	266	dbSNP
rs10028945	271	dbSNP
rs1276847559	273	dbSNP
rs751183559	282	dbSNP
rs1345896389	283	dbSNP
rs1031465102	289	dbSNP
rs755405631	292	dbSNP
rs967665858	299	dbSNP
rs3832300	301	dbSNP
rs993076759	309	dbSNP
rs1409971412	312	dbSNP
rs1374338665	317	dbSNP
rs372181109	325	dbSNP
rs925641482	329	dbSNP
rs935638604	341	dbSNP
rs970397924	349	dbSNP
rs984076959	354	dbSNP
rs1471797505	356	dbSNP
rs1414924213	364	dbSNP
rs1298323744	368	dbSNP
rs1485597184	369	dbSNP
rs1235810128	372	dbSNP
rs1212280640	374	dbSNP
rs1443237080	376	dbSNP
rs147193608	379	dbSNP
rs1216348506	384	dbSNP
rs1290199411	385	dbSNP
rs1326806752	385	dbSNP
rs1228938376	387	dbSNP
rs1276251022	393	dbSNP
rs1300596825	394	dbSNP
rs1346143004	397	dbSNP
rs933698757	405	dbSNP
rs1317191714	409	dbSNP
rs1359421845	409	dbSNP
rs1277090594	410	dbSNP
rs1391309432	410	dbSNP
rs1491505846	410	dbSNP
rs1180682917	411	dbSNP
rs1209071008	411	dbSNP
rs1241810152	411	dbSNP
rs1269822795	411	dbSNP
rs1321104226	411	dbSNP
rs1378578012	411	dbSNP
rs1466371476	411	dbSNP
rs1491579356	411	dbSNP
rs59682924	411	dbSNP
rs1356511530	412	dbSNP
rs1218020899	413	dbSNP
rs1310447794	417	dbSNP
rs1229602614	421	dbSNP
rs1262509728	422	dbSNP
rs1379249583	422	dbSNP
rs562251554	423	dbSNP
rs1201193688	424	dbSNP
rs886903018	425	dbSNP
rs11731834	428	dbSNP
rs1348684133	428	dbSNP
rs1491044276	429	dbSNP
rs1248857360	430	dbSNP
rs1451587645	430	dbSNP
rs1491060750	430	dbSNP
rs909787910	430	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.