>VPS28|NM_183057|3'UTR
   1 GCTGGACGACTCACAGGTGCGTCAGATGCTGTTCGACCTGGAGTCAGCCTACAACGCCTTCAACCGCTTCCTGCATGCCT
  81 GAGCCCGGGGCACTAGCCCTTGCACAGAAGGGCAGAGTCTGAGGCGATGGCTCCTGGTCCCCTGTCCGCCACACAGGCCG
 161 TGGTCATCCACACAACTCACTGTCTGCAGCTGCCTGTCTGGTGTCTGTCTTTGGTGTCAGAACTTTGGGGGCCGGGCCCC
 241 TCCCCACAATAAAGATGCTCTCCGACCTTCCTGCTTGGGTGGCTCCAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cuACGA-GAGACAUG-AGUGUGUu 5' || | | |||| | |||||| Target 5' ccTGGTCCCCTGTCCGCCACACAg 3'	133 - 156	128.00	-13.90
2	miRNA 3' cuaCGAGAGACAUGAGUGUGUu 5' || || | ||::|||| Target 5' gggGCACT-AGCCCTTGCACAg 3'	87 - 107	118.00	-10.00
3	miRNA 3' cuacgagagacaUGAGUGUguu 5' ||||||| Target 5' ----gctggacgACTCACAggt 3'	1 - 18	110.00	-12.60

Mutant ID	Mutant Position	Mutant Source
COSM8604575	1	COSMIC
COSM5793595	20	COSMIC
COSM7180484	21	COSMIC
COSM6536699	25	COSMIC
COSM7817465	25	COSMIC
COSM7150240	34	COSMIC
COSM7973767	40	COSMIC
COSM8100260	45	COSMIC
COSM8601386	46	COSMIC
COSM8625219	55	COSMIC
COSM6516849	56	COSMIC
COSM4503949	67	COSMIC
COSM5467372	70	COSMIC
COSM6820533	74	COSMIC
COSM7821892	75	COSMIC
COSM7661239	81	COSMIC
COSN30526321	86	COSMIC
COSN30450842	87	COSMIC
COSN29631958	89	COSMIC
COSN2525825	99	COSMIC
COSN30180275	115	COSMIC
COSN30457862	125	COSMIC
COSN20076389	127	COSMIC
COSN27004701	129	COSMIC
COSN26547944	133	COSMIC
COSN30461830	139	COSMIC
COSN17177850	147	COSMIC
COSN27004700	159	COSMIC
COSN30682614	160	COSMIC
COSN30153947	188	COSMIC
COSN31512864	234	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs781947127	5	dbSNP
rs781988369	7	dbSNP
rs781931731	8	dbSNP
rs782403965	13	dbSNP
rs782293340	20	dbSNP
rs782625320	31	dbSNP
rs140103128	34	dbSNP
rs1399401432	36	dbSNP
rs939256309	37	dbSNP
rs1294748592	38	dbSNP
rs782633289	46	dbSNP
rs368501757	55	dbSNP
rs781809209	63	dbSNP
rs375225604	65	dbSNP
rs1342003916	66	dbSNP
rs782556531	67	dbSNP
rs1317801785	70	dbSNP
rs781860283	75	dbSNP
rs760551691	84	dbSNP
rs10527	85	dbSNP
rs376242912	86	dbSNP
rs11558160	87	dbSNP
rs781787964	90	dbSNP
rs782726132	91	dbSNP
rs11558161	93	dbSNP
rs782028836	94	dbSNP
rs545259517	96	dbSNP
rs374698280	97	dbSNP
rs782012319	97	dbSNP
rs782362007	99	dbSNP
rs371271081	104	dbSNP
rs1291301232	105	dbSNP
rs781963698	106	dbSNP
rs782367676	113	dbSNP
rs782187435	117	dbSNP
rs368968346	119	dbSNP
rs116517218	125	dbSNP
rs782294422	126	dbSNP
rs782641635	127	dbSNP
rs782522312	131	dbSNP
rs11948	132	dbSNP
rs556590841	140	dbSNP
rs1315510953	141	dbSNP
rs968926432	142	dbSNP
rs537967115	147	dbSNP
rs1010655511	148	dbSNP
rs782274158	154	dbSNP
rs894851641	155	dbSNP
rs567607459	159	dbSNP
rs1217689041	164	dbSNP
rs1033377474	165	dbSNP
rs1003209561	173	dbSNP
rs752498443	175	dbSNP
rs1183386131	179	dbSNP
rs1414365286	180	dbSNP
rs1423196704	182	dbSNP
rs555342032	182	dbSNP
rs1047667985	184	dbSNP
rs534218543	185	dbSNP
rs1381667627	186	dbSNP
rs1419903498	194	dbSNP
rs1297259158	196	dbSNP
rs1360281459	201	dbSNP
rs1428975295	201	dbSNP
rs899017121	203	dbSNP
rs1037899835	211	dbSNP
rs1380483617	213	dbSNP
rs1396005464	223	dbSNP
rs1312988153	224	dbSNP
rs1356022570	226	dbSNP
rs943475880	231	dbSNP
rs767298157	233	dbSNP
rs566864502	234	dbSNP
rs1211915200	239	dbSNP
rs1237872415	242	dbSNP
rs1482868351	243	dbSNP
rs1181729588	249	dbSNP
rs1051738126	255	dbSNP
rs933309398	263	dbSNP
rs551543616	264	dbSNP
rs1157529992	266	dbSNP
rs114205096	267	dbSNP
rs188244036	269	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.