ID	Duplex structure	Position	Score	MFE
1	miRNA 3' guggggGUCUCGGAG--UCGAGGGgu 5' ||||||: | :|||||| Target 5' -----gCAGAGCTGCCAGGCTCCCag 3'	1 - 21	144.00	-23.30
2	miRNA 3' guGGGGGUCUCGGAGUCGAGGGGu 5' || |: |||| |||||| Target 5' ggCCACTTAAGCC----CTCCCCa 3'	312 - 331	127.00	-21.10
3	miRNA 3' guGGGGGUC--UCGGA-GUCGAGGGGu 5' ||:|| | |||| || ||:|| Target 5' gtCCTCCTGCCTGCCTGAAGGTCTCCc 3'	173 - 199	116.00	-24.10

Mutant ID	Mutant Position	Mutant Source
COSN32053460	4	COSMIC
COSN20234418	18	COSMIC
COSN30183358	24	COSMIC
COSN20078801	30	COSMIC
COSN20644203	51	COSMIC
COSN30171582	52	COSMIC
COSN26976841	57	COSMIC
COSN22258339	60	COSMIC
COSN31610117	288	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs764002549	4	dbSNP
rs1363406953	7	dbSNP
rs755095380	11	dbSNP
rs751808023	13	dbSNP
rs1254992103	14	dbSNP
rs1363675559	15	dbSNP
rs1465216836	18	dbSNP
rs912294356	22	dbSNP
rs1421180706	24	dbSNP
rs1404033034	25	dbSNP
rs1409186837	28	dbSNP
rs1199218346	29	dbSNP
rs766360236	33	dbSNP
rs763147438	36	dbSNP
rs563310643	37	dbSNP
rs375957187	38	dbSNP
rs776926799	39	dbSNP
rs769071697	40	dbSNP
rs1318720346	41	dbSNP
rs542987056	42	dbSNP
rs986457818	44	dbSNP
rs1222716681	48	dbSNP
rs201254119	49	dbSNP
rs199627207	50	dbSNP
rs1204572910	62	dbSNP
rs1045436188	81	dbSNP
rs953711835	82	dbSNP
rs948388115	83	dbSNP
rs1456802033	89	dbSNP
rs1179518478	91	dbSNP
rs532745165	92	dbSNP
rs921194348	94	dbSNP
rs755880819	100	dbSNP
rs974363261	109	dbSNP
rs1430991413	113	dbSNP
rs1470046070	114	dbSNP
rs117079025	117	dbSNP
rs541317216	128	dbSNP
rs1448874536	132	dbSNP
rs1298701145	133	dbSNP
rs572648132	138	dbSNP
rs950808944	145	dbSNP
rs73992400	146	dbSNP
rs1251854197	149	dbSNP
rs1325926165	150	dbSNP
rs1224398824	151	dbSNP
rs1013629273	153	dbSNP
rs895106443	160	dbSNP
rs1207188750	166	dbSNP
rs1233966447	167	dbSNP
rs1487838762	172	dbSNP
rs1188933802	176	dbSNP
rs534387064	178	dbSNP
rs1480541007	184	dbSNP
rs1192547000	193	dbSNP
rs1421144646	204	dbSNP
rs911800772	205	dbSNP
rs1316479574	207	dbSNP
rs542322476	214	dbSNP
rs1349216470	216	dbSNP
rs1456391383	217	dbSNP
rs1452925929	219	dbSNP
rs1291831580	223	dbSNP
rs148010496	224	dbSNP
rs143428396	228	dbSNP
rs1277842408	231	dbSNP
rs1338809684	232	dbSNP
rs539873045	233	dbSNP
rs1216699713	238	dbSNP
rs1274215977	239	dbSNP
rs755185706	250	dbSNP
rs1042972701	257	dbSNP
rs945534960	262	dbSNP
rs368651464	264	dbSNP
rs748679205	265	dbSNP
rs1192430286	268	dbSNP
rs1335401247	272	dbSNP
rs781635894	276	dbSNP
rs932327602	277	dbSNP
rs906823124	278	dbSNP
rs1421966969	280	dbSNP
rs920900936	284	dbSNP
rs1024267476	285	dbSNP
rs974037226	286	dbSNP
rs1367490361	288	dbSNP
rs962975498	292	dbSNP
rs1297754734	293	dbSNP
rs1343638720	296	dbSNP
rs908444716	298	dbSNP
rs1271103936	301	dbSNP
rs1378961571	303	dbSNP
rs1227055504	307	dbSNP
rs1236647904	311	dbSNP
rs755090968	313	dbSNP
rs1180709348	315	dbSNP
rs577299938	316	dbSNP
rs182721113	327	dbSNP
rs534866782	330	dbSNP
rs1448342072	332	dbSNP
rs1054229776	341	dbSNP
rs1236634447	344	dbSNP
rs1250908129	345	dbSNP
rs1209194195	347	dbSNP
rs894408	348	dbSNP
rs902926176	356	dbSNP
rs1041564921	357	dbSNP
rs971214374	360	dbSNP
rs370868164	362	dbSNP
rs944538150	367	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.