ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uuUAAU-CCAUCACCGUCACCUu 5' |||| ||| || | ||| | Target 5' aaATTACAGTATTGTCTGTGAAg 3'	141 - 163	104.00	-5.80
2	miRNA 3' uuuaauccaucaccguCACCUu 5' ||||| Target 5' ctgttgatgtaatgatGTGGAa 3'	110 - 131	100.00	-8.40
3	miRNA 3' uuUAAUCCAUCAC-CGUCAccuu 5' | || |:||| |||| Target 5' aaACTA--TGGTGCTCAGTtttg 3'	70 - 90	92.00	-5.76

Mutant ID	Mutant Position	Mutant Source
COSN19707453	7	COSMIC
COSN31599363	25	COSMIC
COSN26642981	30	COSMIC
COSN20051078	35	COSMIC
COSN30156040	45	COSMIC
COSN30477550	46	COSMIC
COSN30189948	48	COSMIC
COSN5087217	59	COSMIC
COSN30517455	81	COSMIC
COSN30482986	92	COSMIC
COSN30157219	94	COSMIC
COSN31516465	94	COSMIC
COSN30532241	129	COSMIC
COSN31610034	142	COSMIC
COSN31602309	145	COSMIC
COSN31586849	156	COSMIC
COSN30126712	163	COSMIC
COSN31539629	171	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs777062028	2	dbSNP
rs1252088773	4	dbSNP
rs764492313	5	dbSNP
rs759149588	6	dbSNP
rs766895377	9	dbSNP
rs1397798395	11	dbSNP
rs773699055	12	dbSNP
rs770665341	16	dbSNP
rs762463562	18	dbSNP
rs760987108	19	dbSNP
rs956741889	20	dbSNP
rs773170649	21	dbSNP
rs1432943135	22	dbSNP
rs1309515144	30	dbSNP
rs769656196	35	dbSNP
rs1324165071	36	dbSNP
rs1412040819	37	dbSNP
rs72843326	38	dbSNP
rs781346126	39	dbSNP
rs1277042627	40	dbSNP
rs768447032	40	dbSNP
rs1262495570	43	dbSNP
rs1168505608	45	dbSNP
rs375083268	49	dbSNP
rs562932953	55	dbSNP
rs1003032383	56	dbSNP
rs1273018772	59	dbSNP
rs969477381	61	dbSNP
rs1168000696	64	dbSNP
rs1044651891	65	dbSNP
rs1477387363	68	dbSNP
rs1429230661	85	dbSNP
rs145215507	90	dbSNP
rs1481646522	94	dbSNP
rs1240156252	95	dbSNP
rs1475003382	96	dbSNP
rs898507927	101	dbSNP
rs1420258461	104	dbSNP
rs1038351981	106	dbSNP
rs3205007	107	dbSNP
rs3205008	109	dbSNP
rs1162928094	110	dbSNP
rs1270046583	114	dbSNP
rs1394023142	116	dbSNP
rs749283603	123	dbSNP
rs1436295732	125	dbSNP
rs1430597906	127	dbSNP
rs540952596	127	dbSNP
rs1196476950	138	dbSNP
rs777633833	145	dbSNP
rs933837370	151	dbSNP
rs866944719	154	dbSNP
rs921055081	155	dbSNP
rs772818777	162	dbSNP
rs1281832024	163	dbSNP
rs1446236775	165	dbSNP
rs1281342050	167	dbSNP
rs975362341	174	dbSNP
rs1484689874	179	dbSNP
rs1345973720	180	dbSNP
rs1417269875	190	dbSNP
rs533112511	197	dbSNP
rs747853799	198	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.