>SFXN4|NM_213649|3'UTR
   1 GCGTGAGTTTTAGGTGAATTTATGTGGTTCCTGCTTGAAAACCTTCCCCTCTCCAGGTTCGGTTTAGAGAACTTTGCCAC
  81 AGGTCTTCTGGGGACCCCAGAGGTGTCTGTGCTGACAAGGCGACTTCAGATTCCATACTGAGATCGTTCCCAGGCTGGCG
 161 TCTCTGGGGTTTTTAAGGCTGGCTGGAGAAGACAGTGGGAGGGTGCCCCGTCTGACACCCCTGGGGTTGCTGAGGGAACG
 241 GTTGGAGTGGGGATCGGCCTGCGAAAGGATACTGTGAAATCACTAATTAACTAATAAACCTGTCTCAAGTTGAGGATTTG
 321 AAGAAAGAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uacaUCC---GGAAAUCUAGUGAa 5' ||| |: | | |||||| Target 5' cgaaAGGATACTGTGAAATCACTa 3'	262 - 285	121.00	-9.00
2	miRNA 3' uacauccggaAAUCUAGUGAa 5' | |||||::| Target 5' gattccatacTGAGATCGTTc 3'	129 - 149	115.00	-7.50
3	miRNA 3' uacauccGGAAAUCUAGUGAa 5' || | :|:|::|| Target 5' ctgacacCCCTGGGGTTGCTg 3'	212 - 232	102.00	-5.20

Mutant ID	Mutant Position	Mutant Source
1188485	145	ClinVar
1191001	205	ClinVar
1186555	256	ClinVar
1194864	271	ClinVar
1196188	272	ClinVar
COSN503938	2	COSMIC
COSN9027701	3	COSMIC
COSN8407170	22	COSMIC
COSN30537892	40	COSMIC
COSN510635	43	COSMIC
COSN31539140	89	COSMIC
COSN30140324	110	COSMIC
COSN19640620	129	COSMIC
COSN31578049	166	COSMIC
COSN31557095	263	COSMIC
COSN10060852	276	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs768770142	1	dbSNP
rs141886415	2	dbSNP
rs202015162	3	dbSNP
rs746422677	5	dbSNP
rs781640449	11	dbSNP
rs1348871621	15	dbSNP
rs770902326	18	dbSNP
rs1210363284	22	dbSNP
rs770540663	22	dbSNP
rs1272326364	24	dbSNP
rs746486862	27	dbSNP
rs777280264	28	dbSNP
rs757991868	46	dbSNP
rs1219537854	47	dbSNP
rs1346013845	50	dbSNP
rs1233434762	51	dbSNP
rs1481863984	53	dbSNP
rs1396302728	56	dbSNP
rs988981182	60	dbSNP
rs376186435	61	dbSNP
rs1033094479	66	dbSNP
rs1412049015	67	dbSNP
rs1329951880	68	dbSNP
rs999830828	81	dbSNP
rs1025744338	90	dbSNP
rs964341348	93	dbSNP
rs1276700090	97	dbSNP
rs1343705735	98	dbSNP
rs973022647	100	dbSNP
rs1230157988	106	dbSNP
rs1350822813	110	dbSNP
rs1307166822	116	dbSNP
rs1017516650	121	dbSNP
rs529514261	122	dbSNP
rs1197395387	125	dbSNP
rs1273367929	126	dbSNP
rs567064806	128	dbSNP
rs2275110	129	dbSNP
rs1026078580	135	dbSNP
rs1254370325	140	dbSNP
rs72832455	145	dbSNP
rs376091445	146	dbSNP
rs999923174	158	dbSNP
rs1038851809	159	dbSNP
rs941409002	160	dbSNP
rs564134430	163	dbSNP
rs905843966	165	dbSNP
rs1044697230	167	dbSNP
rs369078851	168	dbSNP
rs917442175	196	dbSNP
rs1473848805	201	dbSNP
rs530493517	203	dbSNP
rs143805954	205	dbSNP
rs1482309499	209	dbSNP
rs925759441	210	dbSNP
rs1367700160	215	dbSNP
rs561377752	216	dbSNP
rs1270577021	218	dbSNP
rs1338461553	222	dbSNP
rs978917994	225	dbSNP
rs3203943	231	dbSNP
rs142281843	234	dbSNP
rs572748734	236	dbSNP
rs749488951	239	dbSNP
rs1275913246	240	dbSNP
rs987364358	242	dbSNP
rs138956577	252	dbSNP
rs1026573156	255	dbSNP
rs115303392	256	dbSNP
rs937331850	257	dbSNP
rs927407725	262	dbSNP
rs1226293319	263	dbSNP
rs185242367	271	dbSNP
rs182079269	272	dbSNP
rs1383022559	273	dbSNP
rs1006035541	275	dbSNP
rs112915551	277	dbSNP
rs537859348	279	dbSNP
rs756647429	288	dbSNP
rs1317955200	292	dbSNP
rs1324890959	293	dbSNP
rs1449044911	295	dbSNP
rs1397981156	304	dbSNP
rs918764027	308	dbSNP
rs191496503	323	dbSNP
rs1358860718	329	dbSNP
rs555943076	330	dbSNP
rs1332266125	334	dbSNP
rs775828087	340	dbSNP
rs186202669	343	dbSNP
rs745992675	351	dbSNP
rs1352159545	352	dbSNP
rs1205169771	356	dbSNP
rs896037223	365	dbSNP
rs1053772660	367	dbSNP
rs765482563	368	dbSNP
rs912792131	371	dbSNP
rs934886636	372	dbSNP
rs567101225	376	dbSNP
rs953042764	384	dbSNP
rs781484799	385	dbSNP
rs1028635265	389	dbSNP
rs376812634	391	dbSNP
rs145133185	392	dbSNP
rs1189903825	400	dbSNP
rs1464974461	403	dbSNP
rs540008075	404	dbSNP
rs1158057406	408	dbSNP
rs1383165122	410	dbSNP
rs1206745992	413	dbSNP
rs1323226400	414	dbSNP
rs1355796880	417	dbSNP
rs1414122242	418	dbSNP
rs1290308891	419	dbSNP
rs999952187	420	dbSNP
rs7897311	421	dbSNP
rs1239209474	425	dbSNP
rs1278439870	427	dbSNP
rs1020153754	430	dbSNP
rs1281536021	435	dbSNP
rs1437311284	442	dbSNP
rs1485572822	445	dbSNP
rs987061551	453	dbSNP
rs140852391	456	dbSNP
rs1420205982	457	dbSNP
rs1181187222	473	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.