>TMPRSS5|NM_030770|3'UTR
   1 GTCCTGCTGTTTCCTCCAGTCTCACTGCACACCACTGCCTCATGCTTCCTGGGGCCTCCAGCAGCTCCACTAATGGAGGA
  81 GAGGCAGTAGCCTCCGACACAGAACGCATGGACCTCCTACTACTGTGTGTGAGGAACAGTCACTACCCACTGGCCAGCCA
 161 CCCAGCCAACAGGTCTCTCCTCTTGGGCCCTGATTTCAGAGTCCTCTTTCTCACTAGAGACTCAATGACAGAAGAGAGGC
 241 TGGGACTTGGTTGGGCATGCTGTGGTTGCTGAGGGATGAGGGGGAGGAGAGAGGTAGGAGCTGGAGATGAAGAGGCTGCT
 321 AGAAGCAGCAGGAAGCCTGCCCTTCTGCCCTCTCCCCTCCCTGCCCCTGTGTGAGTCTTTTGGGAGGGTGCTGGGAGGTG
 401 CCCCCCGTCCCACCTTTTTCCTGTGCTCTAGGTGGGCTAAGTGCCTCCCTAGAGGACTCCATGGCTGAGAGGCTCCTGGG
 481 CAGATGGGGTCAAGGCTGGGCCAGCCCAGATGAAGCCTATGGGAGTCAGGACCCTCTCCACTCTCCCTCTCCACTCCCCT
 561 TCCTGTTCTCACCTGGCTGTGGCTGGCCCTGTGTGGGGTGGGTACACTGGAAAACAAGAAGGTTGGAGTTGGTCTAGGAC
 641 ATTGGTTTTAAATGACAGTTCTGTGAACTGGTCCAAGGAGTTCTGTTATTAAAGTGATATATGGTCTTGGTCCAAAAAAA
 721 AAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ugcaaauuuaaaGUGGGGAAGa 5' | ||||||| Target 5' tctccctctccaCTCCCCTTCc 3'	542 - 563	142.00	-12.60
2	miRNA 3' ugcaaauUUAAAGU----GGGGAAGa 5' :|||||| ||:|||: Target 5' gggccctGATTTCAGAGTCCTCTTTc 3'	185 - 210	125.00	-10.70
3	miRNA 3' ugcaaaUUUAAAGUGGGGAAga 5' |::| || ||:||| Target 5' gccaacAGGTCTCTCCTCTTgg 3'	165 - 186	120.00	-8.50

Mutant ID	Mutant Position	Mutant Source
1179359	137	ClinVar
COSN31491170	23	COSMIC
COSN30126302	95	COSMIC
COSN6986094	105	COSMIC
COSN30160735	212	COSMIC
COSN20184934	227	COSMIC
COSN31559635	234	COSMIC
COSN6986093	709	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs758992928	18	dbSNP
rs1266861397	22	dbSNP
rs1210317856	23	dbSNP
rs1221766370	25	dbSNP
rs774055582	31	dbSNP
rs371377044	34	dbSNP
rs796197507	34	dbSNP
rs770677868	36	dbSNP
rs749017479	50	dbSNP
rs1002475921	53	dbSNP
rs1249666836	54	dbSNP
rs1419480383	68	dbSNP
rs986027595	70	dbSNP
rs1415584030	72	dbSNP
rs1433692654	74	dbSNP
rs1428655965	79	dbSNP
rs377205552	87	dbSNP
rs954689684	90	dbSNP
rs116205432	92	dbSNP
rs547367520	95	dbSNP
rs1373189511	96	dbSNP
rs1181810092	105	dbSNP
rs1480184934	106	dbSNP
rs1249445863	107	dbSNP
rs1369121388	109	dbSNP
rs961784386	119	dbSNP
rs1279521533	121	dbSNP
rs1212124733	124	dbSNP
rs945133826	126	dbSNP
rs1016029990	132	dbSNP
rs17115826	137	dbSNP
rs1200360297	138	dbSNP
rs1250893848	142	dbSNP
rs139161036	154	dbSNP
rs1023205652	171	dbSNP
rs545275655	173	dbSNP
rs1169095294	174	dbSNP
rs926220175	179	dbSNP
rs985714731	181	dbSNP
rs1333861955	182	dbSNP
rs932865269	192	dbSNP
rs1371586737	200	dbSNP
rs1392737863	201	dbSNP
rs531556352	202	dbSNP
rs1305008739	204	dbSNP
rs922813231	208	dbSNP
rs1449259650	226	dbSNP
rs1369953997	230	dbSNP
rs895963977	231	dbSNP
rs1294917221	232	dbSNP
rs1405911719	234	dbSNP
rs976878229	249	dbSNP
rs1271123256	251	dbSNP
rs1458879960	252	dbSNP
rs961461231	255	dbSNP
rs1221977796	269	dbSNP
rs1057339661	271	dbSNP
rs934826904	273	dbSNP
rs1412879861	280	dbSNP
rs1015678006	281	dbSNP
rs1158618483	282	dbSNP
rs903240003	283	dbSNP
rs1043133478	295	dbSNP
rs1324586744	300	dbSNP
rs1351887637	304	dbSNP
rs947365756	313	dbSNP
rs910574851	314	dbSNP
rs986004560	315	dbSNP
rs933294100	326	dbSNP
rs1291397135	330	dbSNP
rs1334030502	334	dbSNP
rs1417610315	336	dbSNP
rs1403406007	337	dbSNP
rs923186147	338	dbSNP
rs971891164	340	dbSNP
rs774544253	345	dbSNP
rs1465844275	347	dbSNP
rs952731126	350	dbSNP
rs984563916	359	dbSNP
rs146829461	363	dbSNP
rs367575682	363	dbSNP
rs1034068678	365	dbSNP
rs1013581593	366	dbSNP
rs1164259310	369	dbSNP
rs1201758819	374	dbSNP
rs1330593663	386	dbSNP
rs1001123003	399	dbSNP
rs1361366357	403	dbSNP
rs1002146014	406	dbSNP
rs906405170	407	dbSNP
rs1354804231	434	dbSNP
rs1237847962	436	dbSNP
rs769209859	441	dbSNP
rs1370921584	444	dbSNP
rs1009349556	449	dbSNP
rs1286329953	451	dbSNP
rs892256986	455	dbSNP
rs1053448015	462	dbSNP
rs1355368377	471	dbSNP
rs936368584	472	dbSNP
rs562892547	473	dbSNP
rs888798108	475	dbSNP
rs1043103264	480	dbSNP
rs1188860258	481	dbSNP
rs1382579435	486	dbSNP
rs1425417860	489	dbSNP
rs1050490108	494	dbSNP
rs932921281	495	dbSNP
rs922810330	505	dbSNP
rs544481854	506	dbSNP
rs1381203310	507	dbSNP
rs1165953046	511	dbSNP
rs1421739331	516	dbSNP
rs976951837	517	dbSNP
rs562665843	519	dbSNP
rs17115824	521	dbSNP
rs574852578	523	dbSNP
rs933328088	534	dbSNP
rs1333871160	538	dbSNP
rs76470159	557	dbSNP
rs984217775	559	dbSNP
rs193296183	563	dbSNP
rs572061965	565	dbSNP
rs759036413	570	dbSNP
rs1302154715	573	dbSNP
rs13606	581	dbSNP
rs970705774	582	dbSNP
rs1417485716	587	dbSNP
rs1445000623	594	dbSNP
rs1004352581	599	dbSNP
rs1024847035	605	dbSNP
rs1238044103	607	dbSNP
rs78936131	611	dbSNP
rs1009377321	615	dbSNP
rs1201624898	629	dbSNP
rs753176964	632	dbSNP
rs1436553825	636	dbSNP
rs892286145	641	dbSNP
rs984529773	642	dbSNP
rs1032104553	643	dbSNP
rs1441854040	652	dbSNP
rs1191995782	653	dbSNP
rs1377231536	656	dbSNP
rs1000636630	658	dbSNP
rs1174362750	669	dbSNP
rs538912894	670	dbSNP
rs1050103401	671	dbSNP
rs1402202384	672	dbSNP
rs1262368167	676	dbSNP
rs539621068	678	dbSNP
rs1484615703	691	dbSNP
rs1266831706	695	dbSNP
rs556697293	698	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.