>SHMT2|NM_001166356|3'UTR
   1 AGGCACCTGGGAAATGAGGCCCACAGACTCAAAGTTACTCTCCTTCCCCCTACCTGGGCCAGTGAAATAGAAAGCCTTTC
  81 TATTTTTTGGTGCGGGAGGGAAGACCTCTCACTTAGGGCAAGAGCCAGGTATAGTCTCCCTTCCCAGAATTTGTAACTGA
 161 GAAGATCTTTTCTTTTTCCTTTTTTTGGTAACAAGACTTAGAAGGAGGGCCCAGGCACTTTCTGTTTGAACCCCTGTCAT
 241 GATCACAGTGTCAGAGACGCGTCCTCTTTCTTGGGGAAGTTGAGGAGTGCCCTTCAGAGCCAGTAGCAGGCAGGGGTGGG
 321 TAGGCACCCTCCTTCCTGTTTTTATCTAATAAAATGCTAACCTGCCCTGAGTTTCCATTACTGTGGGTGGGGTTCCCCTG
 401 GGCCAAACAGTGATTTGTCTCCCTCAATGTGTACACCGCTCCGCTCCCACCACCGCTACCACAAGGACCCCCGGGGCTGC
 481 AGCCTCCTCTTTCTGTCTCTGATCAGAGCCGACACCAGACGTGATTAGCAGGCGCAGCAAATTCAATTTGTTAAATGAAA
 561 TTGTATTTTGCCCA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gaUUCU-GGU-----UCGGA-GGAAGGGa 5' |||| ||| ||:|| ||||||| Target 5' gcAAGAGCCAGGTATAGTCTCCCTTCCCa 3'	118 - 146	164.00	-21.10
2	miRNA 3' gauUCUGG--UUC----GGAGGAAGGGa 5' ||||: ||| :||||||||| Target 5' cacAGACTCAAAGTTACTCTCCTTCCCc 3'	22 - 49	163.00	-20.90
3	miRNA 3' gauUCUGGUUCGGAGGAAGGGa 5' ||:| | |||||||||: Target 5' ggtAGGC--ACCCTCCTTCCTg 3'	319 - 338	147.00	-19.50

Mutant ID	Mutant Position	Mutant Source
COSN30519060	41	COSMIC
COSN26602638	57	COSMIC
COSN30156193	61	COSMIC
COSN28192288	174	COSMIC
COSN20071288	187	COSMIC
COSN26775754	328	COSMIC
COSN25638802	348	COSMIC
COSN31552472	400	COSMIC
COSN26550605	496	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1318716703	3	dbSNP
rs776322364	4	dbSNP
rs1267305023	6	dbSNP
rs761489843	7	dbSNP
rs369547769	18	dbSNP
rs1254924476	19	dbSNP
rs773049410	22	dbSNP
rs1190742792	24	dbSNP
rs1367457277	25	dbSNP
rs368073830	26	dbSNP
rs762750160	27	dbSNP
rs1342090515	31	dbSNP
rs766301585	32	dbSNP
rs1157370152	33	dbSNP
rs751474653	38	dbSNP
rs1461605333	41	dbSNP
rs1166656200	46	dbSNP
rs1018318623	47	dbSNP
rs868141399	73	dbSNP
rs569698983	76	dbSNP
rs777491878	77	dbSNP
rs746445562	78	dbSNP
rs11557170	81	dbSNP
rs11557169	83	dbSNP
rs770457966	85	dbSNP
rs1349703625	90	dbSNP
rs1301672690	91	dbSNP
rs1432231994	94	dbSNP
rs962469471	95	dbSNP
rs1167417327	103	dbSNP
rs185235043	117	dbSNP
rs980485613	119	dbSNP
rs1426176394	120	dbSNP
rs926314060	128	dbSNP
rs1426849309	130	dbSNP
rs1422073935	139	dbSNP
rs1465231327	140	dbSNP
rs1477491373	141	dbSNP
rs1266585034	146	dbSNP
rs572752383	147	dbSNP
rs1206961908	150	dbSNP
rs939006357	159	dbSNP
rs11557168	173	dbSNP
rs972752823	175	dbSNP
rs923443104	180	dbSNP
rs1464737819	181	dbSNP
rs772967471	197	dbSNP
rs933524360	198	dbSNP
rs991754457	203	dbSNP
rs554647678	210	dbSNP
rs1302444705	211	dbSNP
rs945246287	212	dbSNP
rs1378232659	218	dbSNP
rs371194878	226	dbSNP
rs1281785347	227	dbSNP
rs1280091630	229	dbSNP
rs925107890	231	dbSNP
rs1411987372	232	dbSNP
rs776714750	234	dbSNP
rs936407301	245	dbSNP
rs1263217684	246	dbSNP
rs749771372	248	dbSNP
rs1360459587	255	dbSNP
rs1053420994	256	dbSNP
rs577090328	259	dbSNP
rs540821542	260	dbSNP
rs1009468237	261	dbSNP
rs1486423468	262	dbSNP
rs1219246947	264	dbSNP
rs145812788	265	dbSNP
rs899890870	267	dbSNP
rs1484191461	272	dbSNP
rs1257891329	275	dbSNP
rs1210836421	283	dbSNP
rs1323095475	284	dbSNP
rs1261449150	285	dbSNP
rs1477789051	288	dbSNP
rs1230642157	289	dbSNP
rs1403652259	296	dbSNP
rs1351537189	297	dbSNP
rs1306694631	302	dbSNP
rs1390229967	308	dbSNP
rs750569476	313	dbSNP
rs1001960181	319	dbSNP
rs1033478512	326	dbSNP
rs770776806	329	dbSNP
rs1451226721	330	dbSNP
rs1200699686	332	dbSNP
rs1403469170	344	dbSNP
rs1160991741	347	dbSNP
rs958761699	347	dbSNP
rs998206255	349	dbSNP
rs1390514568	356	dbSNP
rs1051497716	358	dbSNP
rs112143664	367	dbSNP
rs1156651100	371	dbSNP
rs887376101	378	dbSNP
rs769245450	384	dbSNP
rs1256690368	386	dbSNP
rs1182548070	387	dbSNP
rs553143014	391	dbSNP
rs1241328922	392	dbSNP
rs572197786	398	dbSNP
rs1017142056	403	dbSNP
rs1431486942	408	dbSNP
rs1175670234	417	dbSNP
rs1330074337	424	dbSNP
rs371580524	428	dbSNP
rs1454170815	436	dbSNP
rs1324064925	437	dbSNP
rs375353676	438	dbSNP
rs369375949	439	dbSNP
rs1033401544	440	dbSNP
rs1435432987	441	dbSNP
rs373185705	443	dbSNP
rs376265132	443	dbSNP
rs1442663463	444	dbSNP
rs543812804	444	dbSNP
rs913566895	445	dbSNP
rs1190530959	447	dbSNP
rs189601930	447	dbSNP
rs1358209456	448	dbSNP
rs1465520597	449	dbSNP
rs913522359	455	dbSNP
rs373864153	456	dbSNP
rs1046073972	471	dbSNP
rs979742682	473	dbSNP
rs925139124	474	dbSNP
rs1299189392	478	dbSNP
rs532841107	480	dbSNP
rs1054853277	487	dbSNP
rs1488145922	492	dbSNP
rs547631718	493	dbSNP
rs888306068	495	dbSNP
rs1223913575	496	dbSNP
rs921353806	506	dbSNP
rs1170602535	511	dbSNP
rs759410807	512	dbSNP
rs1191355376	516	dbSNP
rs1372556033	516	dbSNP
rs1435126785	517	dbSNP
rs1268568645	518	dbSNP
rs1222137009	519	dbSNP
rs773600442	522	dbSNP
rs1016797966	523	dbSNP
rs1277344669	528	dbSNP
rs560038393	529	dbSNP
rs529795181	534	dbSNP
rs548342053	535	dbSNP
rs954918365	538	dbSNP
rs986409676	545	dbSNP
rs766549553	547	dbSNP
rs1402833121	548	dbSNP
rs574267940	557	dbSNP
rs1308615323	563	dbSNP
rs1157035895	564	dbSNP
rs1395948767	566	dbSNP
rs958232321	574	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.