ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cgUCU---UCGU-AAA----GGUGUGUg 5' ||| |||| ||| ||||||| Target 5' ctAGATACAGCAGTTTATACCCACACAc 3'	92 - 119	156.00	-18.30
2	miRNA 3' cgucuucguaaaGGUGUGUg 5' ||:| || Target 5' ---------tagCCGCCCAt 3'	1 - 11	92.00	-6.20
3	miRNA 3' cgucuucguaaaGGUGUGUg 5' |::|||: Target 5' gcaatgctggccCTGCACGc 3'	38 - 57	92.00	-10.20

Mutant ID	Mutant Position	Mutant Source
COSN30115663	6	COSMIC
COSN30459834	6	COSMIC
COSN6532456	7	COSMIC
COSN30546982	27	COSMIC
COSN30447285	33	COSMIC
COSN31562437	58	COSMIC
COSN30740956	83	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1294320785	2	dbSNP
rs751267314	3	dbSNP
rs371250842	6	dbSNP
rs76093031	7	dbSNP
rs966011432	14	dbSNP
rs11552367	16	dbSNP
rs1218093659	16	dbSNP
rs1261332030	23	dbSNP
rs972781924	25	dbSNP
rs766276152	32	dbSNP
rs1317735714	33	dbSNP
rs1468231302	35	dbSNP
rs1277766382	47	dbSNP
rs753637741	48	dbSNP
rs1034069442	49	dbSNP
rs1364304054	52	dbSNP
rs918568756	56	dbSNP
rs111634825	57	dbSNP
rs931425207	61	dbSNP
rs1261516960	64	dbSNP
rs558828276	66	dbSNP
rs1448493860	69	dbSNP
rs575430138	73	dbSNP
rs1395779195	74	dbSNP
rs747762790	75	dbSNP
rs1167762083	77	dbSNP
rs1462518688	79	dbSNP
rs984144550	84	dbSNP
rs909050726	89	dbSNP
rs1185549114	98	dbSNP
rs1475570127	109	dbSNP
rs1243628105	115	dbSNP
rs937809790	136	dbSNP
rs1054884223	140	dbSNP
rs544220122	145	dbSNP
rs949315777	147	dbSNP
rs1467881345	150	dbSNP
rs1253496585	153	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.