ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gacaccuuacagucCCUCCGu 5' ||| || Target 5' acctgctccttcctGGACGCt 3'	24 - 44	88.00	-5.90

Mutant ID	Mutant Position	Mutant Source
COSN30185914	13	COSMIC
COSN26635116	18	COSMIC
COSN26998740	18	COSMIC
COSN2375376	27	COSMIC
COSN20113315	44	COSMIC
COSN8407571	50	COSMIC
COSN30162237	63	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1253087904	1	dbSNP
rs746166687	2	dbSNP
rs772266496	8	dbSNP
rs775628977	13	dbSNP
rs199835269	14	dbSNP
rs1481606410	15	dbSNP
rs1200673174	17	dbSNP
rs536447952	18	dbSNP
rs1423368977	20	dbSNP
rs1477089492	21	dbSNP
rs1173201534	26	dbSNP
rs976948299	27	dbSNP
rs1354561733	31	dbSNP
rs1240502643	33	dbSNP
rs200005737	35	dbSNP
rs1457010735	38	dbSNP
rs371341141	40	dbSNP
rs11268672	41	dbSNP
rs3217068	41	dbSNP
rs770741494	41	dbSNP
rs373986022	44	dbSNP
rs773226997	45	dbSNP
rs762780205	46	dbSNP
rs368883321	48	dbSNP
rs201549625	50	dbSNP
rs1338156256	51	dbSNP
rs879078702	54	dbSNP
rs528073876	56	dbSNP
rs1244805652	58	dbSNP
rs374219311	60	dbSNP
rs1218384767	70	dbSNP
rs1354154752	78	dbSNP
rs573134543	79	dbSNP
rs908507270	82	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.