ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ggAUGCAAGGUA-UCAGAUGGu 5' || || : :| :||:|:|: Target 5' gaTAGGTATAGTGGGTTTGCTt 3'	58 - 79	103.00	-5.90
2	miRNA 3' ggaugcaagGUAUCAGAUGgu 5' ||| |||| | Target 5' agagaccagCAT-GTCTTCag 3'	14 - 33	95.00	-8.30
3	miRNA 3' ggAUGCAAGG-UAUCAGAUGGu 5' ||:| ||| |||| :||:: Target 5' gaTATGGTCCTATAGGTTATTc 3'	444 - 465	95.00	-9.90

Mutant ID	Mutant Position	Mutant Source
COSN30100678	52	COSMIC
COSN26678960	54	COSMIC
COSN30727915	82	COSMIC
COSN18735895	86	COSMIC
COSN28867976	110	COSMIC
COSN29693306	122	COSMIC
COSN7007345	139	COSMIC
COSN20107894	168	COSMIC
COSN6406918	168	COSMIC
COSN19739690	216	COSMIC
COSN27487337	226	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1431333297	2	dbSNP
rs376890449	6	dbSNP
rs1431843967	7	dbSNP
rs1383518794	9	dbSNP
rs1162973534	11	dbSNP
rs757534920	12	dbSNP
rs1396504041	24	dbSNP
rs1256806517	25	dbSNP
rs1336123599	28	dbSNP
rs761189223	29	dbSNP
rs745956222	31	dbSNP
rs769857642	35	dbSNP
rs748376423	36	dbSNP
rs779881232	43	dbSNP
rs938835331	43	dbSNP
rs200593091	45	dbSNP
rs768453219	46	dbSNP
rs774342310	51	dbSNP
rs766839930	54	dbSNP
rs956241043	56	dbSNP
rs1010812256	75	dbSNP
rs562532769	78	dbSNP
rs1321741741	79	dbSNP
rs1315448050	85	dbSNP
rs1390160765	88	dbSNP
rs1350311038	92	dbSNP
rs1308991679	105	dbSNP
rs747171717	107	dbSNP
rs1021923424	114	dbSNP
rs1214052313	132	dbSNP
rs968939811	136	dbSNP
rs974432930	140	dbSNP
rs1248870271	148	dbSNP
rs1042919	152	dbSNP
rs1370266873	158	dbSNP
rs1417215580	158	dbSNP
rs796639820	158	dbSNP
rs866902	158	dbSNP
rs1411012798	165	dbSNP
rs56199078	169	dbSNP
rs1043045891	177	dbSNP
rs1266351524	179	dbSNP
rs1208805978	182	dbSNP
rs1451972764	193	dbSNP
rs1488557950	197	dbSNP
rs1266679071	200	dbSNP
rs1180030688	208	dbSNP
rs1230645633	216	dbSNP
rs1237584350	216	dbSNP
rs1329041934	216	dbSNP
rs56134721	216	dbSNP
rs796772456	216	dbSNP
rs1343962365	217	dbSNP
rs752251049	222	dbSNP
rs1300852860	225	dbSNP
rs551505351	226	dbSNP
rs907585477	227	dbSNP
rs1400081290	228	dbSNP
rs1453098585	239	dbSNP
rs940570916	240	dbSNP
rs1053133742	242	dbSNP
rs1159744288	245	dbSNP
rs892264890	247	dbSNP
rs566548109	248	dbSNP
rs534138602	257	dbSNP
rs997350672	267	dbSNP
rs1433383612	269	dbSNP
rs1268890173	271	dbSNP
rs1461795122	277	dbSNP
rs1464009540	281	dbSNP
rs762432762	283	dbSNP
rs1269334432	287	dbSNP
rs1030617845	288	dbSNP
rs889020270	290	dbSNP
rs1274606039	292	dbSNP
rs1234294129	293	dbSNP
rs771456693	299	dbSNP
rs1037496708	301	dbSNP
rs1333081214	305	dbSNP
rs1007917198	312	dbSNP
rs548994216	314	dbSNP
rs1042927	317	dbSNP
rs947961274	322	dbSNP
rs1399854221	328	dbSNP
rs1044932480	335	dbSNP
rs746099667	338	dbSNP
rs1171745601	346	dbSNP
rs971769309	352	dbSNP
rs1280253774	356	dbSNP
rs143006593	358	dbSNP
rs1190502816	364	dbSNP
rs1448353456	373	dbSNP
rs1026077933	381	dbSNP
rs1191106995	384	dbSNP
rs1003252307	385	dbSNP
rs557053148	391	dbSNP
rs1042929	395	dbSNP
rs993010292	402	dbSNP
rs1292685404	406	dbSNP
rs751264458	410	dbSNP
rs1347510009	416	dbSNP
rs757079923	426	dbSNP
rs371659538	441	dbSNP
rs1227942296	444	dbSNP
rs891870948	449	dbSNP
rs1010431646	453	dbSNP
rs948987682	456	dbSNP
rs979440041	458	dbSNP
rs1437235143	473	dbSNP
rs3177020	487	dbSNP
rs1161293926	492	dbSNP
rs148224418	493	dbSNP
rs1021787831	499	dbSNP
rs925913989	507	dbSNP
rs1396403543	508	dbSNP
rs1404002105	517	dbSNP
rs1166399868	521	dbSNP
rs1428681927	527	dbSNP
rs578075840	528	dbSNP
rs1169971461	533	dbSNP
rs934668868	536	dbSNP
rs1237427467	540	dbSNP
rs1466910154	547	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.