ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cgucGGUCGAGAU-GGGGGCGGu 5' ::||| ||: ||||| || Target 5' tgtcTTAGCACTGCCCCCCTCCc 3'	81 - 103	130.00	-21.80
2	miRNA 3' cgUCGGUCGAGAUGGGGGCGGu 5' ||: : ||| |||||: || Target 5' gaAGTGGCCTCCACCCCTTCCc 3'	124 - 145	120.00	-17.40
3	miRNA 3' cgucGGUC---GAGAUGGGGGCGgu 5' :||| |||| |||:|| Target 5' ctccTCAGGAACTCT--CCCTGCgc 3'	51 - 73	118.00	-16.60

Mutant ID	Mutant Position	Mutant Source
COSN20041808	4	COSMIC
COSN30506387	5	COSMIC
COSN30666408	16	COSMIC
COSN28866014	72	COSMIC
COSN26440283	73	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs779064577	3	dbSNP
rs748164933	4	dbSNP
rs374197908	5	dbSNP
rs778110310	6	dbSNP
rs745707237	8	dbSNP
rs1182186528	9	dbSNP
rs113349002	13	dbSNP
rs1189848233	16	dbSNP
rs1471404046	19	dbSNP
rs1158040425	22	dbSNP
rs774587201	24	dbSNP
rs982693005	24	dbSNP
rs1406133272	26	dbSNP
rs1470646968	27	dbSNP
rs1300591320	29	dbSNP
rs1402048315	31	dbSNP
rs1392480750	32	dbSNP
rs201626537	34	dbSNP
rs1243284504	41	dbSNP
rs1021091573	42	dbSNP
rs1309595907	43	dbSNP
rs762949652	45	dbSNP
rs1227991736	46	dbSNP
rs1284215767	49	dbSNP
rs576536003	50	dbSNP
rs1208054617	52	dbSNP
rs1255507653	53	dbSNP
rs1162207739	57	dbSNP
rs759493651	60	dbSNP
rs1235474159	64	dbSNP
rs549928466	72	dbSNP
rs976572800	73	dbSNP
rs778252399	80	dbSNP
rs569787128	81	dbSNP
rs1460562517	86	dbSNP
rs12420561	89	dbSNP
rs1166669938	94	dbSNP
rs1471463012	94	dbSNP
rs538927747	96	dbSNP
rs934573680	97	dbSNP
rs1374564754	98	dbSNP
rs145995596	100	dbSNP
rs565983838	102	dbSNP
rs1219533071	103	dbSNP
rs1468110509	108	dbSNP
rs114067690	116	dbSNP
rs1377692201	120	dbSNP
rs1228001190	123	dbSNP
rs1327448384	123	dbSNP
rs925938556	124	dbSNP
rs1287966185	125	dbSNP
rs1376214466	127	dbSNP
rs1272546896	130	dbSNP
rs934956350	132	dbSNP
rs1323944275	143	dbSNP
rs1394616743	144	dbSNP
rs1327417777	145	dbSNP
rs1455372830	152	dbSNP
rs1053294326	156	dbSNP
rs1159618240	165	dbSNP
rs773078343	174	dbSNP
rs1222772735	184	dbSNP
rs1420787595	187	dbSNP
rs1422918364	193	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.