>RPL30|NM_000989|3'UTR
   1 ACCTTTTCACCTACAAAATTTCACCTGCAAACCTTAAACCTGCAAAATTTTCCTTTAATAAAATTTGCTTGTTTTAAAAA
  81 CATTGTATCTAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' acUUU--ACGUUUGGACGUUUUGa 5' ||| | :||||||||||||: Target 5' gcAAACCT-TAAACCTGCAAAATt 3'	27 - 49	157.00	-17.00
2	miRNA 3' acuuuacguuUGGACGUUUUGa 5' |||| |||||: Target 5' --accttttcACCTACAAAATt 3'	1 - 20	112.00	-8.40

Mutant ID	Mutant Position	Mutant Source
COSN26644095	8	COSMIC
COSN23017249	39	COSMIC
COSN30117141	65	COSMIC
COSN30141585	65	COSMIC
COSN30518058	71	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs780648509	9	dbSNP
rs1438725463	11	dbSNP
rs6571	11	dbSNP
rs751157811	13	dbSNP
rs766263100	14	dbSNP
rs1472872617	15	dbSNP
rs1804312	16	dbSNP
rs768258234	20	dbSNP
rs1804311	21	dbSNP
rs748608037	22	dbSNP
rs1248562947	23	dbSNP
rs1222504746	24	dbSNP
rs1414200852	25	dbSNP
rs1354694322	27	dbSNP
rs1207657196	35	dbSNP
rs7004887	39	dbSNP
rs1047363047	43	dbSNP
rs112651906	44	dbSNP
rs776893366	45	dbSNP
rs764250126	47	dbSNP
rs761019872	49	dbSNP
rs10635	53	dbSNP
rs201356804	64	dbSNP
rs890738693	76	dbSNP
rs113405761	81	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.