>KIF2C|NM_006845|3'UTR
   1 CGACTGCAAATAAAAATCTGTTTGGTTTGACACCCAGCCTCTTCCCTGGCCCTCCCCAGAGAACTTTGGGTACCTGGTGG
  81 GTCTAGGCAGGGTCTGAGCTGGGACAGGTTCTGGTAAATGCCAAGTATGGGGGCATCTGGGCCCAGGGCAGCTGGGGAGG
 161 GGGTCAGAGTGACATGGGACACTCCTTTTCTGTTCCTCAGTTGTCGCCCTCACGAGAGGAAGGAGCTCTTAGTTACCCTT
 241 TTGTGTTGCCCTTCTTTCCATCAAGGGGAATGTTCTCAGCATAGAGCTTTCTCCGCAGCATCCTGCCTGCGTGGACTGGC
 321 TGCTAATGGAGAGCTCCCTGGGGTTGTCCTGGCTCTGGGGAGAGAGACGGAGCCTTTAGTACAGCTATCTGCTGGCTCTA
 401 AACCTTCTACGCCTTTGGGCCGAGCACTGAATGTCTTGTACTTTAAAAAAATGTTTCTGAGACCTCTTTCTACTTTACTG
 481 TCTCCCTAGAGATCCTAGAGGATCCCTACTGTTTTCTGTTTTATGTGTTTATACATTGTATGTAACAATAAAGAGAAAAA
 561 ATAAATCAGCTGTTTAAGTGTGTGGAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gagUGUGUGAC--AG-AGGGACCu 5' |||| | | || ||||||| Target 5' ttgACACCCAGCCTCTTCCCTGGc 3'	27 - 50	156.00	-16.80
2	miRNA 3' gagugugugacaGAGGGACCu 5' |||||||| Target 5' gctaatggagagCTCCCTGGg 3'	322 - 342	145.00	-17.41
3	miRNA 3' gaguguGUGACAGAGGGACCu 5' :||||||||||| | Target 5' ctacttTACTGTCTCCCTAGa 3'	470 - 490	139.00	-23.00

Mutant ID	Mutant Position	Mutant Source
COSN30527354	12	COSMIC
COSN30154857	27	COSMIC
COSN30464676	52	COSMIC
COSN508910	54	COSMIC
COSN30106157	71	COSMIC
COSN30106158	72	COSMIC
COSN21571190	570	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs142478220	2	dbSNP
rs149328964	3	dbSNP
rs1162090060	5	dbSNP
rs1393997346	8	dbSNP
rs1415477674	9	dbSNP
rs1003935256	14	dbSNP
rs1472947255	17	dbSNP
rs761740158	17	dbSNP
rs775470664	18	dbSNP
rs1454315294	23	dbSNP
rs1299198673	24	dbSNP
rs578096112	25	dbSNP
rs1340231682	33	dbSNP
rs1398412982	40	dbSNP
rs765323551	44	dbSNP
rs760795559	45	dbSNP
rs1244219203	47	dbSNP
rs764146031	49	dbSNP
rs1354241952	51	dbSNP
rs1270590410	54	dbSNP
rs1041303794	58	dbSNP
rs945576058	64	dbSNP
rs540857768	67	dbSNP
rs1220115507	75	dbSNP
rs1042572554	77	dbSNP
rs996756719	78	dbSNP
rs369041587	80	dbSNP
rs1028187783	84	dbSNP
rs1428723690	91	dbSNP
rs968571838	93	dbSNP
rs1186081308	104	dbSNP
rs1321288079	108	dbSNP
rs575741072	117	dbSNP
rs1055150517	126	dbSNP
rs1406342027	129	dbSNP
rs1166718894	131	dbSNP
rs1392164872	131	dbSNP
rs183873013	133	dbSNP
rs1405692256	145	dbSNP
rs1158788156	157	dbSNP
rs895121126	170	dbSNP
rs1450928321	172	dbSNP
rs1343997967	177	dbSNP
rs1422045802	189	dbSNP
rs1298347704	196	dbSNP
rs1354274736	197	dbSNP
rs1192426521	203	dbSNP
rs1468464039	206	dbSNP
rs574101499	207	dbSNP
rs1322628398	209	dbSNP
rs375333448	214	dbSNP
rs1001276080	215	dbSNP
rs1379390417	222	dbSNP
rs1223044780	224	dbSNP
rs376075863	224	dbSNP
rs1440239701	232	dbSNP
rs993743656	233	dbSNP
rs542998610	237	dbSNP
rs1304574876	243	dbSNP
rs1032320470	249	dbSNP
rs1353648335	251	dbSNP
rs1026063619	252	dbSNP
rs954642248	259	dbSNP
rs987326763	262	dbSNP
rs773490964	263	dbSNP
rs1265884308	272	dbSNP
rs1451986562	272	dbSNP
rs1279437516	277	dbSNP
rs1489087501	278	dbSNP
rs1232736295	281	dbSNP
rs910451581	285	dbSNP
rs964761493	286	dbSNP
rs114533015	289	dbSNP
rs989152585	293	dbSNP
rs115551033	295	dbSNP
rs1177046828	296	dbSNP
rs545302112	304	dbSNP
rs1306007596	306	dbSNP
rs765006926	307	dbSNP
rs1042584978	311	dbSNP
rs928087209	312	dbSNP
rs939599900	314	dbSNP
rs1159795069	322	dbSNP
rs1378914029	323	dbSNP
rs565212067	327	dbSNP
rs555385628	339	dbSNP
rs1407870373	344	dbSNP
rs981056531	345	dbSNP
rs371377994	358	dbSNP
rs1359433723	360	dbSNP
rs1484218916	361	dbSNP
rs926873194	362	dbSNP
rs931544261	365	dbSNP
rs984213569	367	dbSNP
rs1257874841	369	dbSNP
rs1006007559	373	dbSNP
rs1277627345	377	dbSNP
rs1038336468	380	dbSNP
rs1316292065	381	dbSNP
rs1286335472	383	dbSNP
rs1230001706	392	dbSNP
rs897240613	396	dbSNP
rs138385810	397	dbSNP
rs908187126	397	dbSNP
rs939787672	399	dbSNP
rs1203284555	404	dbSNP
rs1240531968	408	dbSNP
rs547590594	411	dbSNP
rs567201248	422	dbSNP
rs1452376631	423	dbSNP
rs1345595729	442	dbSNP
rs901045167	446	dbSNP
rs932494260	450	dbSNP
rs1280809836	462	dbSNP
rs1049511708	464	dbSNP
rs529804604	465	dbSNP
rs1187639095	471	dbSNP
rs146660314	473	dbSNP
rs569303777	477	dbSNP
rs1008672445	481	dbSNP
rs185919266	485	dbSNP
rs892448874	487	dbSNP
rs1231336634	495	dbSNP
rs867665398	502	dbSNP
rs1210576367	519	dbSNP
rs1329767463	523	dbSNP
rs540592005	524	dbSNP
rs764063285	525	dbSNP
rs1242314097	526	dbSNP
rs1179604645	528	dbSNP
rs1395051154	529	dbSNP
rs1312942951	536	dbSNP
rs1381166341	544	dbSNP
rs1362323042	552	dbSNP
rs1321960110	556	dbSNP
rs1459347418	557	dbSNP
rs1453096421	562	dbSNP
rs558017427	565	dbSNP
rs1025056921	567	dbSNP
rs1456675209	570	dbSNP
rs140246765	571	dbSNP
rs1425260154	573	dbSNP
rs534106416	574	dbSNP
rs980940510	589	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.