>FXR2|NM_004860|3'UTR
   1 AACCTCCAACCTGCACCCCTCCCTTCTCCATCTCGCTTGCTGCCCAACACCATGGCCCTCACAGGCCCAACTGACCTGCG
  81 CTGGAGCTGCTCTTATCTAGGGGGGAGGGGGGTGGCACAGCAGCTTGGGTACCCCCCAACCTCCAGGAGCTAGTGGAGGG
 161 GTGTGTAACAGGGTCATACCCCCTCCCTCTTGTCCACCCTACCCCCAGGGTAAGGGGAGCCTCTCTCCTTCCCCATCAGA
 241 CTGGATGTGCCTTTATCCTCTAATGCCCCAATCTCTCTCTGAACACCCCCATTCTCCACCTGTTGGTGGGGGGTGCTCCT
 321 CGACCCACCCAGATTTGACCGTTCAGGGGGCCTCCCCTGCTATCCCTCCTCCCATCCTGTACCCCCCATTTCTGGGGCCT
 401 CATCACTGTGGAAGACGGGGATAGTAAGAGATAAGTGGGTGGGAGGCACGGGGAAGGTTTTGGAGTAGAACCAGGGGTGT
 481 GTATGAAGGGGGGTGACAAGGTCCCCCTGGGGAGGGGACCAACCTTGTCTGGTGGATGAGAAGGCGTATTTATTTTTCAC
 561 TGTACAGTATTTAAAAAGAGAATAAAAAAATCCAAATGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' acccuGAGG-UACGGGGUUc 5' |||: ||||||||| Target 5' ttatcCTCTAATGCCCCAAt 3'	253 - 272	158.00	-16.90
2	miRNA 3' acCCUGAGGUACGGGGUUc 5' ||:: || |||||| Target 5' ttGGGTACC---CCCCAAc 3'	125 - 140	121.00	-13.60
3	miRNA 3' acCCUGAGGU-ACGGGGUUc 5' || | :|| | ||||| Target 5' atGGCCCTCACAGGCCCAAc 3'	52 - 71	117.00	-13.84

Mutant ID	Mutant Position	Mutant Source
COSN30110024	6	COSMIC
COSN518957	18	COSMIC
COSN15618829	36	COSMIC
COSN30109209	39	COSMIC
COSN26743061	40	COSMIC
COSN24393362	59	COSMIC
COSN30523285	73	COSMIC
COSN31586278	80	COSMIC
COSN26677248	150	COSMIC
COSN31483937	181	COSMIC
COSN31606824	202	COSMIC
COSN22397869	249	COSMIC
COSN23015248	344	COSMIC
COSN193543	404	COSMIC
COSN31551629	502	COSMIC
COSN32056071	506	COSMIC
COSN193542	558	COSMIC
COSN31522915	599	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs777787552	3	dbSNP
rs1215437653	4	dbSNP
rs751897191	9	dbSNP
rs1287230681	10	dbSNP
rs1430590319	12	dbSNP
rs964637128	14	dbSNP
rs1305275629	16	dbSNP
rs780435818	18	dbSNP
rs1423130541	21	dbSNP
rs1250906907	23	dbSNP
rs200224563	24	dbSNP
rs550664652	25	dbSNP
rs1162527008	26	dbSNP
rs750555216	29	dbSNP
rs1420991875	35	dbSNP
rs765500745	44	dbSNP
rs1422754014	48	dbSNP
rs374532252	50	dbSNP
rs1441376005	51	dbSNP
rs1369845858	52	dbSNP
rs887643255	58	dbSNP
rs1306548430	62	dbSNP
rs1254746132	65	dbSNP
rs1026294308	79	dbSNP
rs972089999	80	dbSNP
rs963264566	87	dbSNP
rs1395556386	88	dbSNP
rs1313623874	90	dbSNP
rs1206613804	96	dbSNP
rs1016209850	101	dbSNP
rs1273641399	105	dbSNP
rs1342673840	106	dbSNP
rs1207159146	109	dbSNP
rs187608139	111	dbSNP
rs1181952922	113	dbSNP
rs1056478916	118	dbSNP
rs1447279347	128	dbSNP
rs1273737432	129	dbSNP
rs778572420	130	dbSNP
rs953201437	133	dbSNP
rs756682446	153	dbSNP
rs528984923	154	dbSNP
rs1173113196	155	dbSNP
rs926882751	159	dbSNP
rs997272661	161	dbSNP
rs1465555897	164	dbSNP
rs902955245	173	dbSNP
rs1043841793	181	dbSNP
rs1358199772	183	dbSNP
rs1297176938	185	dbSNP
rs1433126386	185	dbSNP
rs1041353838	186	dbSNP
rs78125130	187	dbSNP
rs914112706	189	dbSNP
rs988948747	191	dbSNP
rs539846771	192	dbSNP
rs955845015	198	dbSNP
rs1210818532	199	dbSNP
rs1257212623	202	dbSNP
rs894020953	203	dbSNP
rs922901515	207	dbSNP
rs1248290969	209	dbSNP
rs1450404691	228	dbSNP
rs1309971454	232	dbSNP
rs1423577417	233	dbSNP
rs1414392290	234	dbSNP
rs1410441064	235	dbSNP
rs1408332575	241	dbSNP
rs1455743571	260	dbSNP
rs975792361	264	dbSNP
rs1389680197	267	dbSNP
rs1053971570	271	dbSNP
rs1272855749	273	dbSNP
rs938299081	281	dbSNP
rs1219716194	295	dbSNP
rs1303345856	296	dbSNP
rs926806706	304	dbSNP
rs1326414214	305	dbSNP
rs1006180153	308	dbSNP
rs951840609	309	dbSNP
rs1407501773	310	dbSNP
rs1047022175	312	dbSNP
rs949485849	313	dbSNP
rs1477931974	318	dbSNP
rs919350834	321	dbSNP
rs1432093218	322	dbSNP
rs1471620186	322	dbSNP
rs993959212	335	dbSNP
rs1415153550	340	dbSNP
rs532565520	341	dbSNP
rs972037438	341	dbSNP
rs963377425	344	dbSNP
rs896585215	345	dbSNP
rs1436968259	352	dbSNP
rs909095164	357	dbSNP
rs1348569845	373	dbSNP
rs1004007403	374	dbSNP
rs1048119	392	dbSNP
rs774207388	416	dbSNP
rs528030864	417	dbSNP
rs1226233468	418	dbSNP
rs1288745342	427	dbSNP
rs1048131	428	dbSNP
rs1002170983	439	dbSNP
rs953460029	442	dbSNP
rs905237486	445	dbSNP
rs1030147190	449	dbSNP
rs997771497	450	dbSNP
rs867123244	453	dbSNP
rs1182142667	454	dbSNP
rs946994304	454	dbSNP
rs892623431	458	dbSNP
rs1471213536	460	dbSNP
rs1161637297	463	dbSNP
rs967128982	476	dbSNP
rs1413351554	479	dbSNP
rs748993481	481	dbSNP
rs1011163188	483	dbSNP
rs560584630	489	dbSNP
rs1290362841	491	dbSNP
rs1227491735	493	dbSNP
rs1409099095	494	dbSNP
rs892794922	494	dbSNP
rs1308608109	503	dbSNP
rs1336962780	504	dbSNP
rs1332969254	508	dbSNP
rs1290386923	521	dbSNP
rs777340236	522	dbSNP
rs1283182735	527	dbSNP
rs1356602397	536	dbSNP
rs545587425	545	dbSNP
rs976270495	546	dbSNP
rs866743018	560	dbSNP
rs1340545006	573	dbSNP
rs577788423	580	dbSNP
rs756240046	582	dbSNP
rs183985449	584	dbSNP
rs755759540	589	dbSNP
rs943012259	591	dbSNP
rs1185525562	592	dbSNP
rs1046532361	593	dbSNP
rs113595529	601	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.