ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ugcAGUGACGCAAGAGAGGGGc 5' || || | | |||:||| Target 5' ggaTCTCTTCCTGATCTTCCCt 3'	202 - 223	139.00	-15.59
2	miRNA 3' ugcAGUGACGCAAG-AGAGGGGc 5' || | | | | |:||||| Target 5' gccTCCCCACCTCCATTTCCCCt 3'	320 - 342	131.00	-11.20
3	miRNA 3' ugcaGUGACGCAAGAGAGGGGc 5' ||:| | ||:||| ||| Target 5' cttgCATT-CATTTTCTACCCt 3'	286 - 306	129.00	-13.20

Mutant ID	Mutant Position	Mutant Source
COSN511200	1	COSMIC
COSN30139601	14	COSMIC
COSN30614841	14	COSMIC
COSN30469337	28	COSMIC
COSN26999131	43	COSMIC
COSN30462616	44	COSMIC
COSN30160387	45	COSMIC
COSN26999130	63	COSMIC
COSN31525460	71	COSMIC
COSN7983674	89	COSMIC
COSN30135303	129	COSMIC
COSN31599647	146	COSMIC
COSN30527518	166	COSMIC
COSN32065138	238	COSMIC
COSN7199137	347	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs199727080	10	dbSNP
rs1433013101	20	dbSNP
rs151238335	21	dbSNP
rs1362663405	26	dbSNP
rs773391999	28	dbSNP
rs772089533	29	dbSNP
rs1329505795	35	dbSNP
rs762933245	39	dbSNP
rs1173897147	46	dbSNP
rs940279710	52	dbSNP
rs1467109013	54	dbSNP
rs1291782969	56	dbSNP
rs887346624	72	dbSNP
rs1036000623	80	dbSNP
rs1370070023	81	dbSNP
rs772678796	83	dbSNP
rs906189587	97	dbSNP
rs1326570314	101	dbSNP
rs931433577	110	dbSNP
rs199577492	115	dbSNP
rs185457648	126	dbSNP
rs1386607196	130	dbSNP
rs1217635811	143	dbSNP
rs947956938	147	dbSNP
rs372994426	151	dbSNP
rs991504131	152	dbSNP
rs938700681	159	dbSNP
rs1401463978	162	dbSNP
rs1264255045	165	dbSNP
rs1488454735	169	dbSNP
rs539551661	170	dbSNP
rs557171706	173	dbSNP
rs989677492	174	dbSNP
rs1193288968	183	dbSNP
rs1424422297	189	dbSNP
rs1254173330	193	dbSNP
rs1162571477	194	dbSNP
rs1349179085	199	dbSNP
rs1458373798	200	dbSNP
rs538069347	208	dbSNP
rs977378554	211	dbSNP
rs966684632	216	dbSNP
rs192900771	220	dbSNP
rs1398991122	222	dbSNP
rs1021290173	223	dbSNP
rs1278513532	225	dbSNP
rs772588576	228	dbSNP
rs748595389	229	dbSNP
rs1245716406	234	dbSNP
rs924720586	252	dbSNP
rs1226073254	260	dbSNP
rs142284266	261	dbSNP
rs1214479642	263	dbSNP
rs958078643	267	dbSNP
rs977487849	268	dbSNP
rs1294386843	281	dbSNP
rs1487551577	286	dbSNP
rs535379914	288	dbSNP
rs1216124652	290	dbSNP
rs536113807	292	dbSNP
rs1019357403	300	dbSNP
rs996574884	304	dbSNP
rs900905203	314	dbSNP
rs1424263394	324	dbSNP
rs1346952184	329	dbSNP
rs1304038114	331	dbSNP
rs546199250	346	dbSNP
rs1378243055	347	dbSNP
rs1418926211	351	dbSNP
rs1298609367	353	dbSNP
rs1362023294	365	dbSNP
rs1362981918	372	dbSNP
rs1019320479	375	dbSNP
rs1379339705	387	dbSNP
rs12028064	389	dbSNP
rs1317826805	395	dbSNP
rs45462698	396	dbSNP
rs1431576526	397	dbSNP
rs1347124608	401	dbSNP
rs1027841493	404	dbSNP
rs1448888042	405	dbSNP
rs757819514	405	dbSNP
rs1208294817	408	dbSNP
rs1236961002	414	dbSNP
rs1483655865	418	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.