ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uacauccGGAAAUCUAGUGAa 5' || || |||||| Target 5' --caccaCCCCTAAATCACTt 3'	1 - 19	122.00	-8.60
2	miRNA 3' uacauccGGAAAUCUAGUGAa 5' :|||| | |||| Target 5' cattcctTCTTT-AAACACTc 3'	50 - 69	89.00	-5.10
3	miRNA 3' uacAUCCGGAAAUCUAGUGAa 5' || ||| ||||: :| Target 5' tttTAAGCC-CAAGATTTTTt 3'	144 - 163	89.00	-5.30

Mutant ID	Mutant Position	Mutant Source
COSN30168709	4	COSMIC
COSN20078120	66	COSMIC
COSN1639515	70	COSMIC
COSN30134914	77	COSMIC
COSN31531919	84	COSMIC
COSN30447950	130	COSMIC
COSN8588396	158	COSMIC
COSN31590643	164	COSMIC
COSN30608908	186	COSMIC
COSN8140352	187	COSMIC
COSN28838135	195	COSMIC
COSN16743853	215	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1301652270	2	dbSNP
rs17112002	3	dbSNP
rs774168347	4	dbSNP
rs1043739629	7	dbSNP
rs1288511432	8	dbSNP
rs12590363	10	dbSNP
rs904741245	14	dbSNP
rs761809761	16	dbSNP
rs942701110	16	dbSNP
rs759225207	19	dbSNP
rs767141318	20	dbSNP
rs1470896360	25	dbSNP
rs752091024	28	dbSNP
rs755638723	33	dbSNP
rs765357238	37	dbSNP
rs1278129567	38	dbSNP
rs764007376	42	dbSNP
rs1485351842	44	dbSNP
rs773464873	47	dbSNP
rs753786698	49	dbSNP
rs193287013	50	dbSNP
rs1282903014	51	dbSNP
rs1211308283	58	dbSNP
rs1436760908	70	dbSNP
rs1179087518	71	dbSNP
rs901275905	83	dbSNP
rs959487488	86	dbSNP
rs1047115775	91	dbSNP
rs1172623581	101	dbSNP
rs1413161887	108	dbSNP
rs1394918746	110	dbSNP
rs1433224653	111	dbSNP
rs1357648711	112	dbSNP
rs1314465553	116	dbSNP
rs1448509420	118	dbSNP
rs1344492447	119	dbSNP
rs1156842287	122	dbSNP
rs1401880520	124	dbSNP
rs573714948	127	dbSNP
rs1022831389	128	dbSNP
rs1474002496	132	dbSNP
rs1005375385	135	dbSNP
rs1182766242	137	dbSNP
rs1491542509	137	dbSNP
rs1491462675	138	dbSNP
rs1391275168	141	dbSNP
rs1326945243	148	dbSNP
rs540658996	152	dbSNP
rs376939375	154	dbSNP
rs756927677	158	dbSNP
rs1272594567	159	dbSNP
rs1231584626	161	dbSNP
rs923950459	167	dbSNP
rs1001883961	173	dbSNP
rs1228607447	176	dbSNP
rs1803119	180	dbSNP
rs955582331	185	dbSNP
rs1034713911	186	dbSNP
rs1049498	186	dbSNP
rs1389335436	186	dbSNP
rs3189662	187	dbSNP
rs987929416	196	dbSNP
rs185469522	202	dbSNP
rs1419489100	206	dbSNP
rs561523887	210	dbSNP
rs1338690446	212	dbSNP
rs1427621314	217	dbSNP
rs546055483	217	dbSNP
rs1417809810	220	dbSNP
rs913647663	224	dbSNP
rs564462170	236	dbSNP
rs780926903	239	dbSNP
rs967960316	253	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.