ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ggccAGGCAUGU---UUGAGACGUc 5' ||| | || ||||||| Target 5' aactTCCCT-CATTTCTCTCTGCAa 3'	4 - 27	141.00	-10.40
2	miRNA 3' ggCCAGGCAUGUUUGAGACGUc 5' ||||| | | :|||||| Target 5' taGGTCC-TCC---TTCTGCAc 3'	196 - 213	136.00	-13.20
3	miRNA 3' ggccaggcaUGUUUGAGACguc 5' || |::|||| Target 5' ataggagacACCAGTTCTGact 3'	220 - 241	97.00	-7.02

Mutant ID	Mutant Position	Mutant Source
rs1317973022	1	dbSNP
rs150594317	3	dbSNP
rs781963503	17	dbSNP
rs782274205	33	dbSNP
rs782323971	34	dbSNP
rs781921002	38	dbSNP
rs1160039915	46	dbSNP
rs782099121	48	dbSNP
rs1460939108	58	dbSNP
rs1412323182	59	dbSNP
rs112741092	61	dbSNP
rs1063508	65	dbSNP
rs1063509	66	dbSNP
rs1473366878	69	dbSNP
rs782115783	69	dbSNP
rs1372856008	77	dbSNP
rs1187009180	103	dbSNP
rs1020286667	105	dbSNP
rs1249464171	106	dbSNP
rs1802970	110	dbSNP
rs1450517547	118	dbSNP
rs1287693377	124	dbSNP
rs782439634	125	dbSNP
rs1356890620	130	dbSNP
rs782805564	141	dbSNP
rs1029563871	142	dbSNP
rs1367646949	157	dbSNP
rs1321435498	173	dbSNP
rs1440311189	175	dbSNP
rs781785328	180	dbSNP
rs11539259	182	dbSNP
rs1348196311	186	dbSNP
rs1326185658	188	dbSNP
rs782448663	189	dbSNP
rs1406479258	192	dbSNP
rs1396324440	198	dbSNP
rs1802971	202	dbSNP
rs1167137924	205	dbSNP
rs953596637	208	dbSNP
rs1451667666	213	dbSNP
rs1391686341	223	dbSNP
rs782751362	231	dbSNP
rs1016540890	233	dbSNP
rs1477448139	241	dbSNP
rs1244490475	252	dbSNP
rs1223041274	256	dbSNP
rs961673768	282	dbSNP
rs372772986	284	dbSNP
rs1273762438	296	dbSNP
rs1202818230	311	dbSNP
rs782536375	336	dbSNP
rs1298691497	339	dbSNP
rs1218850543	345	dbSNP
rs1802968	352	dbSNP
rs782433320	359	dbSNP
rs1276699824	367	dbSNP
rs782664738	375	dbSNP
rs782593590	399	dbSNP
rs949142965	401	dbSNP
rs1432061769	416	dbSNP
rs980569247	421	dbSNP
rs926425414	424	dbSNP
rs782788904	435	dbSNP
rs1413558728	443	dbSNP
rs1422569915	450	dbSNP
rs370032163	468	dbSNP
rs1478056509	476	dbSNP
rs1251038424	478	dbSNP
rs782207533	502	dbSNP
rs782350537	504	dbSNP
rs1275103536	531	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.