ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cgagACGAGUCUUGAUGUGAg 5' ||:|: :|||||| Target 5' atggTGTTT----TTACACTa 3'	341 - 357	127.00	-9.60
2	miRNA 3' cgagacgagucuUGAUGUGag 5' ||||||: Target 5' ----------acACTACATgt 3'	1 - 11	109.00	-6.84
3	miRNA 3' cgagacgagucuuGAUGUGAg 5' :||:||| Target 5' atgtggatatttcTTATACTa 3'	372 - 392	108.00	-5.90

Mutant ID	Mutant Position	Mutant Source
COSN18721347	25	COSMIC
COSN31600106	26	COSMIC
COSN21333447	168	COSMIC
COSN26562715	176	COSMIC
COSN29050194	246	COSMIC
COSN30174180	335	COSMIC
COSN26316373	394	COSMIC
COSN31563870	410	COSMIC
COSN26464941	453	COSMIC
COSN31515371	491	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1268869746	5	dbSNP
rs1281121256	7	dbSNP
rs774163938	7	dbSNP
rs1488293367	8	dbSNP
rs1191954959	9	dbSNP
rs754017807	10	dbSNP
rs1269567113	11	dbSNP
rs192209386	13	dbSNP
rs1040061418	15	dbSNP
rs751300438	22	dbSNP
rs754742836	23	dbSNP
rs1421956803	24	dbSNP
rs781057226	26	dbSNP
rs761379629	27	dbSNP
rs370186191	29	dbSNP
rs1294859827	32	dbSNP
rs1191808113	33	dbSNP
rs933816586	33	dbSNP
rs1337551437	36	dbSNP
rs912835299	40	dbSNP
rs369964676	41	dbSNP
rs777253936	45	dbSNP
rs1051827643	51	dbSNP
rs1039886452	57	dbSNP
rs1454593697	60	dbSNP
rs899881298	63	dbSNP
rs936742002	64	dbSNP
rs890559666	65	dbSNP
rs892458689	66	dbSNP
rs1378934656	67	dbSNP
rs1413122572	68	dbSNP
rs1462276767	78	dbSNP
rs1351105543	87	dbSNP
rs564515783	87	dbSNP
rs1262316216	89	dbSNP
rs1009596035	90	dbSNP
rs1052425821	92	dbSNP
rs1351157712	105	dbSNP
rs1352973369	106	dbSNP
rs1227925995	122	dbSNP
rs1315662191	126	dbSNP
rs1454587949	128	dbSNP
rs1375710060	129	dbSNP
rs1333695371	136	dbSNP
rs1383036526	138	dbSNP
rs1283470216	140	dbSNP
rs1020598367	141	dbSNP
rs116174854	149	dbSNP
rs1003087636	155	dbSNP
rs1386337005	156	dbSNP
rs1166021463	172	dbSNP
rs1011148864	173	dbSNP
rs1034192050	194	dbSNP
rs758793492	198	dbSNP
rs973783239	206	dbSNP
rs1447291501	219	dbSNP
rs1049235	223	dbSNP
rs145796477	231	dbSNP
rs1263572383	239	dbSNP
rs1026717807	243	dbSNP
rs1023848571	250	dbSNP
rs1487842422	255	dbSNP
rs1212167229	257	dbSNP
rs533907872	262	dbSNP
rs969487644	264	dbSNP
rs867186165	275	dbSNP
rs1357340981	276	dbSNP
rs1283714915	279	dbSNP
rs982488781	283	dbSNP
rs1256316997	284	dbSNP
rs1328476685	293	dbSNP
rs879202614	304	dbSNP
rs1482821657	308	dbSNP
rs1185300876	315	dbSNP
rs1399708037	319	dbSNP
rs769356520	320	dbSNP
rs112709662	325	dbSNP
rs1305469365	328	dbSNP
rs762091880	332	dbSNP
rs1180749989	349	dbSNP
rs1410994964	354	dbSNP
rs1162194881	360	dbSNP
rs975948346	362	dbSNP
rs1374371891	365	dbSNP
rs145384087	367	dbSNP
rs1487811736	372	dbSNP
rs921434853	373	dbSNP
rs1489858314	374	dbSNP
rs1289223606	389	dbSNP
rs1405184979	392	dbSNP
rs1196393219	394	dbSNP
rs772618416	409	dbSNP
rs982978302	410	dbSNP
rs1218272679	430	dbSNP
rs1177467737	432	dbSNP
rs1285663791	433	dbSNP
rs1358919042	438	dbSNP
rs546504593	440	dbSNP
rs909542817	448	dbSNP
rs1330152396	453	dbSNP
rs962701177	455	dbSNP
rs1398039371	458	dbSNP
rs1168070249	466	dbSNP
rs1477312788	470	dbSNP
rs1429730381	484	dbSNP
rs1201390305	494	dbSNP
rs1476924317	497	dbSNP
rs892571679	507	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.