>KLHDC8B|NM_173546|3'UTR
   1 AGGCTTGGTGGGAGCTGTCCACTGGAGCAGCTCATTGCCAGAGGCAGCTATTTCTATGGCTCCTTTTGCTGCTGAGGACA
  81 CTCACTGTGGCTCTGTGGGATGAGAGAGGCATGGGGGTGAGCACTTGAAACACTGCCTTGGGGCCTTGGGTTAGGGGAGC
 161 CTTTGTCTTTAGTGCAGGACACACATATGCTTACACCTACCTTTATCACCATTCGTTCATGAATCATGCCTAGCTCCATC
 241 CTTGCCCTGGGACCTACTAGGCCTTCCATCCAACTGGGAAATGGGGAGAAGCAAAGCTGGCCTCATGCTCTTCAGGGTCA
 321 GTTCCTATCTGGAGTTGACCAGGCCTACCCCAGTTGCCATTCCTGAAAAATCTCAGCTGCCAGGCTGCCTTTAGGGTCCC
 401 TGCAGACCCAGGAGAGTTGAGAGGGTGGGGGACACAGAGAGAATAGAGAGGATGTGGGAACTGCCAGAGGGCCGGAGCGC
 481 AGGAGTTCAAGTGGAGGAATGCTGGCTTTGAGCCCTCTACACTGCTGGTTGTATGACCTTGGACAAGTCACTTCACCTCT
 561 CTGTGCCTCAGCATCCTCATCTATAAATGGGGATCTCTGAAACCTTCCTACCCTACCTACCTCACAGGGCTGTTGTGAGG
 641 ACCCAGGGAGTTTGGATGTGGAAGTAAAAGTGCTGCTAAAACCTAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' guggUCGAUCGGAGUGg 5' ||||:||||||: Target 5' gcaaAGCTGGCCTCATg 3'	291 - 307	145.00	-19.40
2	miRNA 3' guggucGAU-CGGAGUgg 5' ||: |||||| Target 5' acctctCTGTGCCTCAgc 3'	555 - 572	123.00	-13.90
3	miRNA 3' guggucgaucGGAGUGg 5' |||||| Target 5' accctacctaCCTCACa 3'	610 - 626	120.00	-12.90

Mutant ID	Mutant Position	Mutant Source
COSN30178376	8	COSMIC
COSN30474511	25	COSMIC
COSN31585630	86	COSMIC
COSN6769430	494	COSMIC
rs9586	404	GWAS

Mutant ID	Mutant Position	Mutant Source
rs111352447	1	dbSNP
rs777531678	4	dbSNP
rs1033592433	6	dbSNP
rs749291742	8	dbSNP
rs779074178	10	dbSNP
rs749645818	12	dbSNP
rs770904221	12	dbSNP
rs552639807	17	dbSNP
rs745405025	18	dbSNP
rs1206607945	21	dbSNP
rs956192287	25	dbSNP
rs1470036399	28	dbSNP
rs1407310361	38	dbSNP
rs150542030	42	dbSNP
rs775317976	43	dbSNP
rs372241018	44	dbSNP
rs772093674	47	dbSNP
rs746634447	49	dbSNP
rs770336847	55	dbSNP
rs773704789	57	dbSNP
rs1182582226	60	dbSNP
rs763171639	63	dbSNP
rs766935301	64	dbSNP
rs774685046	68	dbSNP
rs528646226	69	dbSNP
rs767473553	70	dbSNP
rs752976713	75	dbSNP
rs1461570778	80	dbSNP
rs1372535624	81	dbSNP
rs1226481394	83	dbSNP
rs939375113	85	dbSNP
rs756327324	91	dbSNP
rs764233031	92	dbSNP
rs753642301	93	dbSNP
rs1289458041	94	dbSNP
rs1172770304	97	dbSNP
rs756952181	100	dbSNP
rs762130113	102	dbSNP
rs745757543	113	dbSNP
rs1043526007	118	dbSNP
rs767825748	123	dbSNP
rs757971910	124	dbSNP
rs1180623254	127	dbSNP
rs1383928630	128	dbSNP
rs1420525723	134	dbSNP
rs1475759230	137	dbSNP
rs1413162621	138	dbSNP
rs943722709	142	dbSNP
rs547064408	143	dbSNP
rs1348394871	146	dbSNP
rs1401765429	148	dbSNP
rs1162990831	150	dbSNP
rs1159378397	154	dbSNP
rs1455350677	170	dbSNP
rs1007863362	171	dbSNP
rs1018873149	176	dbSNP
rs976367613	180	dbSNP
rs756437301	189	dbSNP
rs921050417	191	dbSNP
rs951762858	193	dbSNP
rs1261930137	195	dbSNP
rs568420759	215	dbSNP
rs139491293	216	dbSNP
rs1246137957	221	dbSNP
rs969587185	228	dbSNP
rs1360757394	241	dbSNP
rs888281107	267	dbSNP
rs939803281	273	dbSNP
rs143176164	278	dbSNP
rs939636439	283	dbSNP
rs1209809028	284	dbSNP
rs895713190	286	dbSNP
rs913931156	291	dbSNP
rs1158088145	301	dbSNP
rs946773158	308	dbSNP
rs1486908274	315	dbSNP
rs1161402220	318	dbSNP
rs1210988850	320	dbSNP
rs1373467905	321	dbSNP
rs1194821752	328	dbSNP
rs1444901978	347	dbSNP
rs1261836679	352	dbSNP
rs778345610	353	dbSNP
rs1187870087	356	dbSNP
rs146662619	372	dbSNP
rs888907284	374	dbSNP
rs905982193	376	dbSNP
rs1227135360	380	dbSNP
rs1356029982	381	dbSNP
rs1271113873	383	dbSNP
rs1230121601	385	dbSNP
rs1338703580	388	dbSNP
rs1297879664	390	dbSNP
rs1040653364	391	dbSNP
rs1367538375	394	dbSNP
rs1321469098	398	dbSNP
rs568896626	401	dbSNP
rs1000690219	403	dbSNP
rs9586	404	dbSNP
rs1396817620	406	dbSNP
rs1476757269	411	dbSNP
rs1422456011	412	dbSNP
rs956220455	422	dbSNP
rs1199717132	424	dbSNP
rs1010337028	430	dbSNP
rs1019176303	432	dbSNP
rs1313622439	436	dbSNP
rs1244498635	442	dbSNP
rs771321870	445	dbSNP
rs966251150	450	dbSNP
rs976420178	451	dbSNP
rs1274400221	459	dbSNP
rs920925517	461	dbSNP
rs953699888	466	dbSNP
rs1277727206	469	dbSNP
rs1316458180	469	dbSNP
rs984287037	470	dbSNP
rs1347421686	473	dbSNP
rs774679387	474	dbSNP
rs1005936169	475	dbSNP
rs1219023703	479	dbSNP
rs1330885434	480	dbSNP
rs1399311015	482	dbSNP
rs1176640702	489	dbSNP
rs939855574	514	dbSNP
rs1470738669	515	dbSNP
rs1022729517	516	dbSNP
rs1299565514	520	dbSNP
rs970245676	522	dbSNP
rs1036847560	525	dbSNP
rs1470247201	532	dbSNP
rs557377423	533	dbSNP
rs1181053392	538	dbSNP
rs1456689542	540	dbSNP
rs1257849439	562	dbSNP
rs11552	563	dbSNP
rs917200118	582	dbSNP
rs572496538	584	dbSNP
rs1204770608	593	dbSNP
rs539800641	610	dbSNP
rs980960474	628	dbSNP
rs1276854563	636	dbSNP
rs745971684	637	dbSNP
rs1284754701	638	dbSNP
rs1035620688	640	dbSNP
rs1281148041	656	dbSNP
rs772132885	665	dbSNP
rs905865626	673	dbSNP
rs1242344947	679	dbSNP
rs1470872153	686	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.