>PKM|NM_002654|3'UTR
   1 TGGACCCCAGAGCCCCTCCTCCAGCCCCTGTCCCACCCCCTTCCCCCAGCCCATCCATTAGGCCAGCAACGCTTGTAGAA
  81 CTCACTCTGGGCTGTAACGTGGCACTGGTAGGTTGGGACACCAGGGAAGAAGATCAACGCCTCACTGAAACATGGCTGTG
 161 TTTGCAGCCTGCTCTAGTGGGACAGCCCAGAGCCTGGCTGCCCATCATGTGGCCCCACCCAATCAAGGGAAGAAGGAGGA
 241 ATGCTGGACTGGAGGCCCCTGGAGCCAGATGGCAAGAGGGTGACAGCTTCCTTTCCTGTGTGTACTCTGTCCAGTTCCTT
 321 TAGAAAAAATGGATGCCCAGAGGACTCCCAACCCTGGCTTGGGGTCAAGAAACAGCCAGCAAGAGTTAGGGGCCTTAGGG
 401 CACTGGGCTGTTGTTCCATTGAAGCCGACTCTGGCCCTGGCCCTTACTTGCTTCTCTAGCTCTCTAGGCCTCTCCAGTTT
 481 GCACCTGTCCCCACCCTCCACTCAGCTGTCCTGCAGCAAACACTCCACCCTCCACCTTCCATTTTCCCCCACTACTGCAG
 561 CACCTCCAGGCCTGTTGCTATAGAGCCTACCTGTATGTCAATAAACAACAGCTGAAGCACCAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cucuaccACCUCAUCGAGGUCCg 5' || || | ||||||| Target 5' ccactacTGCAGCACCTCCAGGc 3'	549 - 571	156.00	-15.00
2	miRNA 3' cucuACCACCUCAUC--GAGGUCCg 5' |||| |: |:| | ||||| Target 5' gcacTGGTAGGTTGGGACACCAGGg 3'	102 - 126	129.00	-14.90
3	miRNA 3' cucuaccaccucAUC--GAGGUCCg 5' ||| |||:||| Target 5' tacttgcttctcTAGCTCTCTAGGc 3'	445 - 469	129.00	-13.40

Mutant ID	Mutant Position	Mutant Source
COSN30461144	22	COSMIC
COSN30625394	33	COSMIC
COSN30472203	40	COSMIC
COSN26989238	43	COSMIC
COSN30474339	47	COSMIC
COSN19693401	49	COSMIC
COSN1179778	72	COSMIC
COSN22825602	149	COSMIC
COSN28857797	158	COSMIC
COSN30642908	201	COSMIC
COSN20078304	208	COSMIC
COSN28559453	252	COSMIC
COSN22070276	290	COSMIC
COSN31547305	323	COSMIC
COSN18834766	528	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1027535730	1	dbSNP
rs554090702	3	dbSNP
rs200738909	8	dbSNP
rs1374368973	10	dbSNP
rs759054927	12	dbSNP
rs1193489103	13	dbSNP
rs1422932565	14	dbSNP
rs1220239119	18	dbSNP
rs11558361	21	dbSNP
rs1253430651	22	dbSNP
rs10732234	28	dbSNP
rs1461703362	30	dbSNP
rs1257363051	33	dbSNP
rs772413973	34	dbSNP
rs1383178324	35	dbSNP
rs11558374	39	dbSNP
rs3202686	40	dbSNP
rs1381481352	41	dbSNP
rs1303527642	48	dbSNP
rs1228397478	52	dbSNP
rs1340139337	54	dbSNP
rs1313004759	57	dbSNP
rs1008328734	58	dbSNP
rs749459822	61	dbSNP
rs756192452	67	dbSNP
rs745908092	68	dbSNP
rs750409915	69	dbSNP
rs1049758804	70	dbSNP
rs564605428	71	dbSNP
rs1168897728	73	dbSNP
rs751410507	76	dbSNP
rs1383065256	77	dbSNP
rs763790054	81	dbSNP
rs1444721900	84	dbSNP
rs1237454640	86	dbSNP
rs1176727215	87	dbSNP
rs758125349	91	dbSNP
rs1250930468	93	dbSNP
rs752407217	94	dbSNP
rs1481493781	96	dbSNP
rs764912493	98	dbSNP
rs893426277	99	dbSNP
rs41277722	103	dbSNP
rs774005711	104	dbSNP
rs776262901	106	dbSNP
rs1019729948	108	dbSNP
rs1399006509	110	dbSNP
rs768113350	111	dbSNP
rs1192082200	112	dbSNP
rs762222704	118	dbSNP
rs1006877448	123	dbSNP
rs774837890	135	dbSNP
rs1386578870	136	dbSNP
rs934856789	137	dbSNP
rs768917291	138	dbSNP
rs867743392	139	dbSNP
rs749589469	140	dbSNP
rs1379128216	141	dbSNP
rs756755982	149	dbSNP
rs1156342332	151	dbSNP
rs567441132	152	dbSNP
rs1460763271	158	dbSNP
rs1471117016	158	dbSNP
rs1241269379	159	dbSNP
rs1233443461	160	dbSNP
rs1200286075	163	dbSNP
rs1318397442	164	dbSNP
rs1250939587	172	dbSNP
rs1225464335	174	dbSNP
rs1197825579	176	dbSNP
rs1449655070	177	dbSNP
rs770828820	192	dbSNP
rs897281063	201	dbSNP
rs1490587284	210	dbSNP
rs1452098301	214	dbSNP
rs8192432	218	dbSNP
rs915613984	219	dbSNP
rs991175643	220	dbSNP
rs11558362	221	dbSNP
rs1447947344	222	dbSNP
rs1378840666	229	dbSNP
rs1353403099	230	dbSNP
rs1280653837	233	dbSNP
rs895539875	244	dbSNP
rs1232939245	248	dbSNP
rs1344051250	251	dbSNP
rs1305392540	252	dbSNP
rs11558372	255	dbSNP
rs1056835900	256	dbSNP
rs1433874968	256	dbSNP
rs1359574859	258	dbSNP
rs775719202	260	dbSNP
rs769952543	262	dbSNP
rs1408535203	266	dbSNP
rs930294090	268	dbSNP
rs1479304255	272	dbSNP
rs1431118480	273	dbSNP
rs1393065771	284	dbSNP
rs1199968045	288	dbSNP
rs1307075415	291	dbSNP
rs1480880921	295	dbSNP
rs11558373	305	dbSNP
rs920545973	306	dbSNP
rs1352686959	311	dbSNP
rs1448846795	314	dbSNP
rs1285518402	318	dbSNP
rs971962406	320	dbSNP
rs1322082545	322	dbSNP
rs1285576120	323	dbSNP
rs1224570426	324	dbSNP
rs1339380706	335	dbSNP
rs182636985	340	dbSNP
rs1268046644	341	dbSNP
rs745997051	343	dbSNP
rs1323681262	353	dbSNP
rs1345291942	354	dbSNP
rs1332982135	361	dbSNP
rs1468166654	363	dbSNP
rs1481033361	365	dbSNP
rs1179440041	373	dbSNP
rs1236923009	385	dbSNP
rs1289181752	391	dbSNP
rs961897225	405	dbSNP
rs1227180338	407	dbSNP
rs1350781516	410	dbSNP
rs549117240	413	dbSNP
rs753524122	415	dbSNP
rs1280645002	420	dbSNP
rs533829630	426	dbSNP
rs926976636	427	dbSNP
rs1463116594	435	dbSNP
rs1329187999	436	dbSNP
rs759030691	438	dbSNP
rs866117510	439	dbSNP
rs1376009677	440	dbSNP
rs1042921204	442	dbSNP
rs566347711	444	dbSNP
rs758754361	445	dbSNP
rs913188951	456	dbSNP
rs987675460	458	dbSNP
rs3209004	462	dbSNP
rs1062430	464	dbSNP
rs955486564	470	dbSNP
rs1028823600	472	dbSNP
rs375147398	482	dbSNP
rs996835095	483	dbSNP
rs1367614444	488	dbSNP
rs1321849297	489	dbSNP
rs1457664470	494	dbSNP
rs1404831912	496	dbSNP
rs552042592	500	dbSNP
rs1385841559	501	dbSNP
rs965269614	513	dbSNP
rs912627179	517	dbSNP
rs746246644	526	dbSNP
rs1180905248	528	dbSNP
rs1411175277	530	dbSNP
rs1429963086	537	dbSNP
rs551403089	543	dbSNP
rs1033245485	546	dbSNP
rs139819709	550	dbSNP
rs1244562402	551	dbSNP
rs1026989486	556	dbSNP
rs1357191748	559	dbSNP
rs903406523	561	dbSNP
rs995545640	563	dbSNP
rs1045909158	578	dbSNP
rs1220446536	579	dbSNP
rs961919890	589	dbSNP
rs1276309595	592	dbSNP
rs950226093	595	dbSNP
rs1015873439	601	dbSNP
rs1281415950	606	dbSNP
rs1441022078	620	dbSNP
rs111336659	622	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.