ID	Duplex structure	Position	Score	MFE
1	miRNA 3' aggggaAGGGACGGUAUAGGAGu 5' ||||| || | |:||| Target 5' tttagcTCCCTTCCTTTTTCTCt 3'	59 - 81	121.00	-17.00
2	miRNA 3' aggggAAGGGACGGUAUAGGAGu 5' ||: :| :: |:|:||| Target 5' ttttaTTTATTTTTTTGTTCTCc 3'	241 - 263	118.00	-5.90
3	miRNA 3' agGGGAAGGGACG---GU----AUAGGAGu 5' | ||| |: |: || |||:||: Target 5' ctCACTTACTCGTAAACATAGGTATTCTTt 3'	365 - 394	115.00	-8.10

Mutant ID	Mutant Position	Mutant Source
COSN31606452	3	COSMIC
COSN30735585	6	COSMIC
COSN26457527	121	COSMIC
COSN28870135	151	COSMIC
COSN22898324	164	COSMIC
COSN24392833	169	COSMIC
COSN29659153	238	COSMIC
COSN32131222	777	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs369707288	3	dbSNP
rs372108102	4	dbSNP
rs906744812	5	dbSNP
rs1299722578	13	dbSNP
rs376710077	27	dbSNP
rs1393089673	28	dbSNP
rs1215423431	31	dbSNP
rs1315826466	33	dbSNP
rs1034351541	37	dbSNP
rs1413803187	37	dbSNP
rs757079299	44	dbSNP
rs1245968744	50	dbSNP
rs1360379784	51	dbSNP
rs1402447065	55	dbSNP
rs1042876	57	dbSNP
rs1324834132	62	dbSNP
rs1364984258	81	dbSNP
rs1388303771	92	dbSNP
rs1301798597	95	dbSNP
rs1173570368	105	dbSNP
rs1306688811	108	dbSNP
rs1393867860	127	dbSNP
rs1190996990	129	dbSNP
rs1450173140	135	dbSNP
rs963918652	143	dbSNP
rs959616834	144	dbSNP
rs1213942469	145	dbSNP
rs1444391329	147	dbSNP
rs1259872772	155	dbSNP
rs994924238	164	dbSNP
rs1209973991	168	dbSNP
rs530175478	169	dbSNP
rs1029365717	170	dbSNP
rs950347857	171	dbSNP
rs955203750	172	dbSNP
rs569588314	189	dbSNP
rs924333161	200	dbSNP
rs1311883533	205	dbSNP
rs935508487	209	dbSNP
rs1373956125	214	dbSNP
rs989567995	215	dbSNP
rs1430899877	218	dbSNP
rs1489548771	222	dbSNP
rs1419396656	223	dbSNP
rs974538870	224	dbSNP
rs921855484	226	dbSNP
rs1238322399	230	dbSNP
rs1191981772	231	dbSNP
rs1261534109	232	dbSNP
rs575376158	236	dbSNP
rs990451799	237	dbSNP
rs1306890031	242	dbSNP
rs566784228	246	dbSNP
rs948362447	248	dbSNP
rs534493317	258	dbSNP
rs915274787	272	dbSNP
rs1045710574	276	dbSNP
rs1427078163	277	dbSNP
rs558951401	293	dbSNP
rs1356368569	301	dbSNP
rs190311175	303	dbSNP
rs906587274	313	dbSNP
rs1353978858	314	dbSNP
rs1047192058	315	dbSNP
rs1310290719	318	dbSNP
rs939530491	323	dbSNP
rs1354536653	331	dbSNP
rs1168934044	335	dbSNP
rs1465704754	335	dbSNP
rs1418578170	341	dbSNP
rs1164513789	348	dbSNP
rs542696794	365	dbSNP
rs538395890	366	dbSNP
rs767129244	368	dbSNP
rs1459172057	372	dbSNP
rs895406826	375	dbSNP
rs556806067	376	dbSNP
rs182809164	377	dbSNP
rs899657903	383	dbSNP
rs996806318	386	dbSNP
rs1280040770	387	dbSNP
rs1029968210	408	dbSNP
rs541972774	410	dbSNP
rs1330820879	432	dbSNP
rs1232756452	445	dbSNP
rs1288579922	450	dbSNP
rs1271415866	471	dbSNP
rs1323562187	487	dbSNP
rs1390370907	488	dbSNP
rs1296958763	489	dbSNP
rs1439165899	504	dbSNP
rs752198595	505	dbSNP
rs553712930	509	dbSNP
rs1325356302	510	dbSNP
rs977455990	516	dbSNP
rs1411871698	541	dbSNP
rs1485112408	552	dbSNP
rs1004549585	555	dbSNP
rs775672738	564	dbSNP
rs1031384025	566	dbSNP
rs1418289358	569	dbSNP
rs571911679	579	dbSNP
rs1475207301	580	dbSNP
rs111731075	581	dbSNP
rs564175765	595	dbSNP
rs1488794501	613	dbSNP
rs1211348753	621	dbSNP
rs1411052852	621	dbSNP
rs1465848638	622	dbSNP
rs201856018	628	dbSNP
rs989849331	635	dbSNP
rs1214543020	637	dbSNP
rs1316725588	639	dbSNP
rs764678963	640	dbSNP
rs865798633	648	dbSNP
rs974997969	670	dbSNP
rs1165074360	679	dbSNP
rs1221268957	680	dbSNP
rs1344188439	684	dbSNP
rs1303299301	691	dbSNP
rs575939302	695	dbSNP
rs543201638	713	dbSNP
rs1289701728	716	dbSNP
rs1453263353	722	dbSNP
rs981065075	726	dbSNP
rs928178423	729	dbSNP
rs939375451	745	dbSNP
rs990117025	751	dbSNP
rs1445596725	755	dbSNP
rs1454544821	764	dbSNP
rs1394460503	769	dbSNP
rs754138836	770	dbSNP
rs1417645525	771	dbSNP
rs561596836	774	dbSNP
rs1450793355	777	dbSNP
rs530114701	784	dbSNP
rs548431168	787	dbSNP
rs1047058441	791	dbSNP
rs1484714629	793	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.