>LMBR1L|NM_018113|3'UTR
   1 CCTCCAGCTGGGGGTGGGAAGGAAAAAACTGGACACTGCCATCTGCTGCCTAGGCCTGGAGGGAAGCCCAAGGCTACTTG
  81 GACCTCAGGACCTGGAATCTGAGAGGGTGGGTGGCAGAGGGGAGCAGAGCCATCTGCACTATTGCATAATCTGAGCCAGA
 161 GTTTGGGACCAGGACCTCCTGCTTTTCCATACTTAACTGTGGCCTCAGCATGGGGTAGGGCTGGGTGACTGGGTCTAGCC
 241 CCTGATCCCAAATCTGTTTACACATCAATCTGCCTCACTGCTGTTCTGGGCCATCCCCATAGCCATGTTTACATGATTTG
 321 ATGTGCAATAGGGTGGGGTAGGGGCAGGGAAAGGACTGGGCCAGGGCAGGCTCGGGAGATAGATTGTCTCCCTTGCCTCT
 401 GGCCCAGCAGAGCCTAAGCACTGTGCTATCCTGGAGGGGCTTTGGACCACCTGAAAGACCAAGGGGATAGGGAGGAGGAG
 481 GCTTCAGCCATCAGCAATAAAGTTGATCCCAGGGTTTGCTTTGTTTTTTTAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cuuUA-GAGAUGUCUUCGGGGUau 5' || ||: || | :|||:|| Target 5' tccATACTTAACTGTGGCCTCAgc 3'	186 - 209	124.00	-11.90
2	miRNA 3' cuUUAG--AGAUGU---CUU---CGGGGUau 5' |||| |:|||| || |||:|| Target 5' caAATCTGTTTACACATCAATCTGCCTCAct 3'	249 - 279	123.00	-10.10
3	miRNA 3' cuuuaGAGAUGUCUUCGGGGUAU 5' :|||: : | ||||||| Target 5' tgctgTTCTGGGCCATCCCCATA 3'	279 - 301	122.00	-14.40

Mutant ID	Mutant Position	Mutant Source
COSN30484367	5	COSMIC
COSN30118848	7	COSMIC
COSN30455612	12	COSMIC
COSN31504034	17	COSMIC
COSN26982625	22	COSMIC
COSN30525459	25	COSMIC
COSN24392967	39	COSMIC
COSN30459780	61	COSMIC
COSN20077643	71	COSMIC
COSN30153356	101	COSMIC
COSN31573480	149	COSMIC
COSN31486057	184	COSMIC
COSN7616448	269	COSMIC
COSN1148788	276	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1358071031	6	dbSNP
rs192672535	7	dbSNP
rs574819296	8	dbSNP
rs371523859	10	dbSNP
rs540826142	13	dbSNP
rs1301237098	14	dbSNP
rs1477533848	26	dbSNP
rs1247227515	29	dbSNP
rs1224608394	34	dbSNP
rs779058769	36	dbSNP
rs1285357757	40	dbSNP
rs1217683145	43	dbSNP
rs754971592	44	dbSNP
rs1299382479	45	dbSNP
rs1281005842	47	dbSNP
rs200146930	49	dbSNP
rs1219347696	54	dbSNP
rs947198968	56	dbSNP
rs1433052773	58	dbSNP
rs1306328742	59	dbSNP
rs1204456123	65	dbSNP
rs915607403	67	dbSNP
rs1233895451	70	dbSNP
rs530644210	74	dbSNP
rs1484258974	84	dbSNP
rs991178880	97	dbSNP
rs1268503746	107	dbSNP
rs1480866047	112	dbSNP
rs538009920	117	dbSNP
rs187490591	122	dbSNP
rs1409384608	125	dbSNP
rs1169697012	127	dbSNP
rs1371039980	129	dbSNP
rs1431456340	139	dbSNP
rs928574556	152	dbSNP
rs1344554949	163	dbSNP
rs763593679	173	dbSNP
rs927537852	182	dbSNP
rs1276196289	189	dbSNP
rs984110052	192	dbSNP
rs931334411	193	dbSNP
rs552872781	197	dbSNP
rs918569709	199	dbSNP
rs1457709865	204	dbSNP
rs1291494720	229	dbSNP
rs1490590854	230	dbSNP
rs973237816	239	dbSNP
rs539365135	240	dbSNP
rs184573212	242	dbSNP
rs546262742	247	dbSNP
rs1475141230	251	dbSNP
rs961927435	257	dbSNP
rs529736311	259	dbSNP
rs567248153	263	dbSNP
rs1032240339	268	dbSNP
rs1000760827	270	dbSNP
rs1405670538	271	dbSNP
rs902193779	274	dbSNP
rs776040235	276	dbSNP
rs952833745	286	dbSNP
rs1028795651	292	dbSNP
rs996874598	298	dbSNP
rs1297650179	305	dbSNP
rs1013792621	306	dbSNP
rs901191720	313	dbSNP
rs192312136	314	dbSNP
rs1211957254	315	dbSNP
rs1205655565	325	dbSNP
rs1487996449	335	dbSNP
rs1019615410	338	dbSNP
rs1446611208	341	dbSNP
rs1263486012	344	dbSNP
rs1247200179	346	dbSNP
rs999143671	361	dbSNP
rs1249966091	363	dbSNP
rs770073769	370	dbSNP
rs1043252248	373	dbSNP
rs530576567	374	dbSNP
rs1473015940	378	dbSNP
rs774339570	380	dbSNP
rs1226267807	384	dbSNP
rs1405392278	394	dbSNP
rs1399747251	408	dbSNP
rs1320708197	421	dbSNP
rs1367516208	423	dbSNP
rs894238847	436	dbSNP
rs1336679642	438	dbSNP
rs1055928843	439	dbSNP
rs938427529	448	dbSNP
rs187794352	460	dbSNP
rs149778606	461	dbSNP
rs1425747126	463	dbSNP
rs931532423	464	dbSNP
rs918588159	468	dbSNP
rs1203060297	472	dbSNP
rs1255445159	474	dbSNP
rs375365067	476	dbSNP
rs1189435687	485	dbSNP
rs1230946226	502	dbSNP
rs1472107022	511	dbSNP
rs762501418	518	dbSNP
rs182856886	525	dbSNP
rs1159447846	531	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.