ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uacaUCCGGAAAUCUAGUGAa 5' ||||: |||:||| Target 5' caaaAGGCT----GATTACTg 3'	445 - 461	131.00	-9.90
2	miRNA 3' uacauccggaaaucUAGUGAa 5' |||||| Target 5' ggaaaattctcttaATCACTa 3'	22 - 42	120.00	-5.70
3	miRNA 3' uacauccggaaaucUAGUGAa 5' :||||| Target 5' cacaggtgacaagcGTCACTt 3'	118 - 138	104.00	-9.40

Mutant ID	Mutant Position	Mutant Source
COSN30146119	5	COSMIC
COSN30113136	31	COSMIC
COSN30448142	33	COSMIC
COSN30155140	39	COSMIC
COSN31492565	50	COSMIC
COSN30534247	61	COSMIC
COSN31496094	80	COSMIC
COSN30582846	86	COSMIC
COSN30135666	185	COSMIC
COSN31482061	199	COSMIC
COSN31541405	219	COSMIC
COSN30175031	250	COSMIC
COSN31962712	273	COSMIC
COSN31563625	274	COSMIC
COSN31516198	328	COSMIC
COSN8795131	419	COSMIC
COSN25399771	441	COSMIC
COSN31520446	471	COSMIC
COSN31571148	476	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs868174237	5	dbSNP
rs1355491281	8	dbSNP
rs777097133	10	dbSNP
rs889769440	26	dbSNP
rs751112486	29	dbSNP
rs756794725	31	dbSNP
rs1386070502	32	dbSNP
rs1185871753	33	dbSNP
rs1224119117	37	dbSNP
rs745579256	40	dbSNP
rs1356088746	47	dbSNP
rs1463819410	48	dbSNP
rs1214603686	53	dbSNP
rs1313633093	61	dbSNP
rs1247735665	68	dbSNP
rs575526698	68	dbSNP
rs1008288595	74	dbSNP
rs1019631065	80	dbSNP
rs961205360	81	dbSNP
rs763781665	82	dbSNP
rs1359788409	83	dbSNP
rs376545581	85	dbSNP
rs906940259	91	dbSNP
rs1336158870	96	dbSNP
rs1225638610	102	dbSNP
rs1240966559	109	dbSNP
rs1253862057	111	dbSNP
rs938455075	116	dbSNP
rs1055622653	122	dbSNP
rs894264020	125	dbSNP
rs1388447527	128	dbSNP
rs1291907764	130	dbSNP
rs753549369	132	dbSNP
rs544298943	133	dbSNP
rs980036916	139	dbSNP
rs1389983367	142	dbSNP
rs1194310195	143	dbSNP
rs370165133	145	dbSNP
rs574442685	148	dbSNP
rs745798792	150	dbSNP
rs1420031263	151	dbSNP
rs1293246135	153	dbSNP
rs1202612347	155	dbSNP
rs1340968896	157	dbSNP
rs992596818	167	dbSNP
rs1229310185	168	dbSNP
rs1393496537	169	dbSNP
rs758226756	171	dbSNP
rs190997894	175	dbSNP
rs1329328958	180	dbSNP
rs1300497524	189	dbSNP
rs1434344406	193	dbSNP
rs912957429	198	dbSNP
rs559937746	199	dbSNP
rs532818541	209	dbSNP
rs1174280021	217	dbSNP
rs1042988871	226	dbSNP
rs1377378236	227	dbSNP
rs1031030707	233	dbSNP
rs1168915727	234	dbSNP
rs1463852562	237	dbSNP
rs780060318	241	dbSNP
rs936946785	242	dbSNP
rs183180977	243	dbSNP
rs754223221	244	dbSNP
rs1179345243	246	dbSNP
rs889800473	248	dbSNP
rs746719943	251	dbSNP
rs562708842	261	dbSNP
rs1018368407	265	dbSNP
rs1008152581	266	dbSNP
rs1301059742	269	dbSNP
rs1019662086	272	dbSNP
rs1369698548	273	dbSNP
rs897209711	274	dbSNP
rs777557326	275	dbSNP
rs531809679	276	dbSNP
rs548236987	277	dbSNP
rs1393306569	288	dbSNP
rs920151826	289	dbSNP
rs536268890	295	dbSNP
rs568206691	298	dbSNP
rs928330709	299	dbSNP
rs1034244964	303	dbSNP
rs938367170	309	dbSNP
rs1471975033	325	dbSNP
rs1232885944	339	dbSNP
rs549724333	343	dbSNP
rs534074731	348	dbSNP
rs1488	349	dbSNP
rs1204037781	352	dbSNP
rs41267839	354	dbSNP
rs1266163798	355	dbSNP
rs1227645368	358	dbSNP
rs945819236	368	dbSNP
rs1453954930	369	dbSNP
rs1241481768	371	dbSNP
rs538892525	374	dbSNP
rs1336537745	375	dbSNP
rs1453819308	379	dbSNP
rs186703774	386	dbSNP
rs575465831	394	dbSNP
rs998810097	398	dbSNP
rs538045159	403	dbSNP
rs936829220	418	dbSNP
rs759694284	428	dbSNP
rs1426788390	430	dbSNP
rs1049978218	458	dbSNP
rs554841760	461	dbSNP
rs911437849	475	dbSNP
rs1184048388	478	dbSNP
rs1355030319	478	dbSNP
rs574580890	479	dbSNP
rs748314394	481	dbSNP
rs767864393	482	dbSNP
rs1245906465	490	dbSNP
rs1463462954	494	dbSNP
rs891088242	497	dbSNP
rs1208058418	509	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.