ID	Duplex structure	Position	Score	MFE
1	miRNA 3' guGAGUCGCC-----GGCA--AAGGGGUu 5' || ||| | :| | |||||:| Target 5' caCTGAGCTGTAACTTCTTCCTTCCCTAt 3'	267 - 295	130.00	-13.80
2	miRNA 3' gugagucgccggcaaAGGGGUu 5' |||||| Target 5' ctgacctgtgctcccTCCCCAa 3'	181 - 202	120.00	-14.60
3	miRNA 3' guGAGUCGCCGGCAAAGGGGuu 5' :|| | ||| |||||: Target 5' tgTTC--CTGCC-CTTCCCTtt 3'	36 - 54	111.00	-9.40

Mutant ID	Mutant Position	Mutant Source
29755	67	ClinVar
COSN26978905	18	COSMIC
COSN30526302	24	COSMIC
COSN30145701	26	COSMIC
COSN17134393	35	COSMIC
COSN20476147	43	COSMIC
COSN19631150	51	COSMIC
COSN26978904	66	COSMIC
COSN17134460	67	COSMIC
COSN20476148	75	COSMIC
COSN20476149	79	COSMIC
COSN30188260	88	COSMIC
COSN31581613	102	COSMIC
COSN15656245	113	COSMIC
COSN1330872	122	COSMIC
COSN1330873	124	COSMIC
COSN19620218	136	COSMIC
COSN28773765	139	COSMIC
COSN30498797	145	COSMIC
COSN19631151	146	COSMIC
COSN19631086	152	COSMIC
COSN30518887	181	COSMIC
COSN15653401	199	COSMIC
COSN20075704	206	COSMIC
COSN28645313	218	COSMIC
COSN27602173	237	COSMIC
COSN6513647	264	COSMIC
rs1061235	67	GWAS

Mutant ID	Mutant Position	Mutant Source
rs1255082501	10	dbSNP
rs1177032289	14	dbSNP
rs1484382958	15	dbSNP
rs752204014	17	dbSNP
rs200105102	20	dbSNP
rs45502194	26	dbSNP
rs1487958414	29	dbSNP
rs1190937442	30	dbSNP
rs1207148423	33	dbSNP
rs1243570672	34	dbSNP
rs1061156	35	dbSNP
rs1168668342	38	dbSNP
rs771413680	42	dbSNP
rs572407231	43	dbSNP
rs149604815	44	dbSNP
rs1376588533	45	dbSNP
rs746262450	51	dbSNP
rs1372238184	52	dbSNP
rs10853	59	dbSNP
rs200062476	63	dbSNP
rs45574131	66	dbSNP
rs1061235	67	dbSNP
rs1444122973	69	dbSNP
rs1357293904	70	dbSNP
rs1451946086	73	dbSNP
rs372982354	75	dbSNP
rs375982247	79	dbSNP
rs1396948741	87	dbSNP
rs1156974002	93	dbSNP
rs1479545179	93	dbSNP
rs1418716379	96	dbSNP
rs769833442	101	dbSNP
rs1409063728	106	dbSNP
rs578027483	112	dbSNP
rs13395	113	dbSNP
rs1469710052	116	dbSNP
rs1157312752	118	dbSNP
rs386698558	121	dbSNP
rs796763843	122	dbSNP
rs796782603	124	dbSNP
rs181754482	134	dbSNP
rs1307263839	135	dbSNP
rs548664078	135	dbSNP
rs113042145	136	dbSNP
rs1353356457	138	dbSNP
rs763522908	141	dbSNP
rs386698559	142	dbSNP
rs1061815	143	dbSNP
rs41540713	144	dbSNP
rs376071102	146	dbSNP
rs547626665	148	dbSNP
rs879755443	149	dbSNP
rs367837921	152	dbSNP
rs1360144630	153	dbSNP
rs41545022	155	dbSNP
rs1289240412	156	dbSNP
rs1417552282	161	dbSNP
rs202133784	165	dbSNP
rs977990404	171	dbSNP
rs923836459	180	dbSNP
rs1197721184	181	dbSNP
rs539771433	194	dbSNP
rs112863520	199	dbSNP
rs1332278838	202	dbSNP
rs186532503	203	dbSNP
rs1233739663	212	dbSNP
rs66585529	218	dbSNP
rs1293360823	220	dbSNP
rs1445709210	221	dbSNP
rs1362721970	223	dbSNP
rs1290748474	229	dbSNP
rs1460065421	230	dbSNP
rs199807456	237	dbSNP
rs1166032175	245	dbSNP
rs1463348170	247	dbSNP
rs79244404	252	dbSNP
rs572546465	253	dbSNP
rs1215076505	255	dbSNP
rs1432044073	262	dbSNP
rs201469499	264	dbSNP
rs144149732	270	dbSNP
rs879593791	278	dbSNP
rs112525507	284	dbSNP
rs145464605	287	dbSNP
rs1252009151	288	dbSNP
rs1382890622	291	dbSNP
rs1245104283	292	dbSNP
rs201615458	295	dbSNP
rs1344009048	305	dbSNP
rs577937205	308	dbSNP
rs1308070709	309	dbSNP
rs2499	311	dbSNP
rs9385	323	dbSNP
rs1370439494	332	dbSNP
rs1194312896	337	dbSNP
rs573809399	357	dbSNP
rs1443507957	368	dbSNP
rs1394757478	395	dbSNP
rs866524175	398	dbSNP
rs2975053	399	dbSNP
rs1171837912	401	dbSNP
rs1452040407	402	dbSNP
rs1386913013	403	dbSNP
rs386698560	406	dbSNP
rs879714036	406	dbSNP
rs13488	407	dbSNP
rs2975052	408	dbSNP
rs370789221	410	dbSNP
rs368263062	411	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.