>TSPAN1|NM_005727|3'UTR
   1 GTCCACTTCTGCCTCTGCCACTACTGCTGCCACATGGGAACTGTGAAGAGGCACCCTGGCAAGCAGCAGTGATTGGGGGA
  81 GGGGACAGGATCTAACAATGTCACTTGGGCCAGAATGGACCTGCCCTTTCTGCTCCAGACTTGGGGCTAGATAGGGACCA
 161 CTCCTTTTAGGCGATGCCTGACTTTCCTTCCATTGGTGGGTGGATGGGTGGGGGGCATTCCAGAGCCTCTAAGGTAGCCA
 241 GTTCTGTTGCCCATTCCCCCAGTCTATTAAACCCTTGATATGCCCCCTAGGCCTAGTGGTGATCCCAGTGCTCTACTGGG
 321 GGATGAGAGAAAGGCATTTTATAGCCTGGGCATAAGTGAAATCAGCAGAGCCTCTGGGTGGATGTGTAGAAGGCACTTCA
 401 AAATGCATAAACCTGTTACAATGTTGCCAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' agacaUUUAUAGGGAAACGGACu 5' |:: ||::: | ||||| Target 5' gagaaAGGCATTTTATAGCCTGg 3'	327 - 349	122.00	-9.20
2	miRNA 3' agacauuuauagggaaACGGACu 5' |||||| Target 5' cactccttttaggcgaTGCCTGa 3'	159 - 181	120.00	-12.71
3	miRNA 3' agaCAUUUAU-AGGGAAACGGacu 5' |||: | |:|: ||||| Target 5' aagGTAGCCAGTTCTGTTGCCcat 3'	231 - 254	116.00	-9.72

Mutant ID	Mutant Position	Mutant Source
COSN27000268	39	COSMIC
COSN27000267	41	COSMIC
COSN23014370	52	COSMIC
COSN31612384	52	COSMIC
COSN30475678	85	COSMIC
COSN31517142	119	COSMIC
COSN30478900	126	COSMIC
COSN16128675	131	COSMIC
COSN31479707	139	COSMIC
COSN30104957	164	COSMIC
COSN32065131	369	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1385327204	2	dbSNP
rs763432931	3	dbSNP
rs769019369	6	dbSNP
rs1162296389	8	dbSNP
rs1197120793	9	dbSNP
rs1489779213	10	dbSNP
rs774960760	13	dbSNP
rs760060391	15	dbSNP
rs199685801	18	dbSNP
rs1319795286	24	dbSNP
rs753427063	26	dbSNP
rs1221004399	31	dbSNP
rs759190086	32	dbSNP
rs1319943806	34	dbSNP
rs1317182350	39	dbSNP
rs1360289464	43	dbSNP
rs1236835755	48	dbSNP
rs929086591	50	dbSNP
rs201843939	51	dbSNP
rs1319090162	52	dbSNP
rs112015052	54	dbSNP
rs890342859	56	dbSNP
rs1444269612	58	dbSNP
rs941445938	59	dbSNP
rs973899735	60	dbSNP
rs1456976655	64	dbSNP
rs1347540895	65	dbSNP
rs865970135	68	dbSNP
rs1419987103	70	dbSNP
rs1379275133	77	dbSNP
rs1177238150	89	dbSNP
rs1802371	92	dbSNP
rs918408766	107	dbSNP
rs1490707132	115	dbSNP
rs1268684578	128	dbSNP
rs373512282	137	dbSNP
rs929867048	143	dbSNP
rs1288196575	145	dbSNP
rs1261811780	146	dbSNP
rs1353308175	153	dbSNP
rs1284141365	160	dbSNP
rs1236687226	165	dbSNP
rs556863534	165	dbSNP
rs1313672333	167	dbSNP
rs1301895681	169	dbSNP
rs1050989202	171	dbSNP
rs944628841	173	dbSNP
rs750243105	174	dbSNP
rs1400077978	178	dbSNP
rs1256810868	191	dbSNP
rs575522016	193	dbSNP
rs1156400033	194	dbSNP
rs1010896826	202	dbSNP
rs542554576	208	dbSNP
rs1378534084	210	dbSNP
rs892844981	210	dbSNP
rs1176006091	211	dbSNP
rs1011747802	212	dbSNP
rs902623329	216	dbSNP
rs1265933341	233	dbSNP
rs1000071848	245	dbSNP
rs1035181556	247	dbSNP
rs1802372	248	dbSNP
rs1261316233	250	dbSNP
rs1185197898	259	dbSNP
rs1202323151	261	dbSNP
rs1346895150	262	dbSNP
rs1802309	274	dbSNP
rs1256318080	275	dbSNP
rs902678577	277	dbSNP
rs1384377686	278	dbSNP
rs1321415258	280	dbSNP
rs11552232	283	dbSNP
rs1158182985	284	dbSNP
rs961183370	294	dbSNP
rs1361236137	301	dbSNP
rs879207176	310	dbSNP
rs1023926464	316	dbSNP
rs561305303	317	dbSNP
rs999671937	318	dbSNP
rs1433504853	321	dbSNP
rs567827047	328	dbSNP
rs960964630	330	dbSNP
rs528452773	334	dbSNP
rs1302673339	338	dbSNP
rs1464853380	347	dbSNP
rs1171937256	353	dbSNP
rs1426483164	353	dbSNP
rs1421517498	354	dbSNP
rs1189903021	376	dbSNP
rs1443259156	381	dbSNP
rs1255734532	383	dbSNP
rs1396937683	384	dbSNP
rs973069641	393	dbSNP
rs920319676	395	dbSNP
rs112046563	415	dbSNP
rs1160698768	418	dbSNP
rs1361219713	420	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.