>ZNF85|NM_003429|3'UTR
   1 AAATTATGGCAAAGCTTTAATCAATTTACAAGTCTTACTAAACATAAGAAAATTTATACTGGAGAGAAACTACTAACCTG
  81 AAAGATGTGACAATAATTTTGACAACACCTCAGACTTATAAAAGTAATCATACTGGTGAGAAATTCTAAAAATGTGAAGA
 161 CTATGGCAAAGTCTTTAAATGGTTGTCACACTTTAGGTAAGATAATTCATATTGGAACAAACTACAAGTGCAAACAATGT
 241 GGCAAAACTTAATTTATGCTCACACCTTACTGCACAGAAAAGAATTTTTAGTTGAGAAAAAGTATACAAATATAAAGAAT
 321 GTGGAAAAGCCGTTAATATCTGCTCACATCTTACTCAGCATCAGAAAGTACTTAATAAAAGCATTATAAATGGAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ggacgaggUUUUUAGGUAa 5' :|||| ||:| Target 5' agaatgtgGAAAAGCCGTt 3'	316 - 334	103.00	-5.40
2	miRNA 3' ggACGAGGUUUUUAGGuaa 5' || | |:||||:| Target 5' acTGGT-GAGAAATTCtaa 3'	132 - 149	96.00	-7.52
3	miRNA 3' ggACGAG---GUUUUUAGGUAa 5' ||| | ||:|||:: | Target 5' acTGCACAGAAAAGAATTTTTa 3'	269 - 290	89.00	-6.92

Mutant ID	Mutant Position	Mutant Source
COSN13791967	27	COSMIC
COSN30145302	76	COSMIC
COSN20115603	119	COSMIC
COSN30565017	121	COSMIC
COSN30177333	152	COSMIC
COSN20115605	178	COSMIC
COSN20115607	196	COSMIC
COSN6139678	227	COSMIC
COSN23268226	324	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1325350502	4	dbSNP
rs370544885	7	dbSNP
rs1464204273	10	dbSNP
rs373664434	12	dbSNP
rs764516013	16	dbSNP
rs547433359	17	dbSNP
rs56137123	22	dbSNP
rs1019401984	25	dbSNP
rs767919394	32	dbSNP
rs201368631	33	dbSNP
rs1259750347	40	dbSNP
rs1478811352	44	dbSNP
rs751001161	47	dbSNP
rs1347304851	49	dbSNP
rs752274992	49	dbSNP
rs983178061	52	dbSNP
rs907583482	54	dbSNP
rs1475088256	56	dbSNP
rs1191602350	61	dbSNP
rs944872860	67	dbSNP
rs1195476866	73	dbSNP
rs988658469	76	dbSNP
rs1209108748	81	dbSNP
rs1255770906	81	dbSNP
rs1040587832	84	dbSNP
rs536152830	87	dbSNP
rs1020142455	89	dbSNP
rs555745788	92	dbSNP
rs970715078	102	dbSNP
rs1338082222	103	dbSNP
rs1466552530	106	dbSNP
rs569525227	110	dbSNP
rs979451254	117	dbSNP
rs59182323	118	dbSNP
rs538097096	119	dbSNP
rs1292323317	120	dbSNP
rs1412642006	125	dbSNP
rs932252365	130	dbSNP
rs938070662	131	dbSNP
rs557910794	132	dbSNP
rs56339351	136	dbSNP
rs1409276029	140	dbSNP
rs903871774	155	dbSNP
rs55637671	162	dbSNP
rs1037310217	163	dbSNP
rs554157883	164	dbSNP
rs1031097584	167	dbSNP
rs1476346735	168	dbSNP
rs897397834	174	dbSNP
rs1181604275	176	dbSNP
rs35802891	176	dbSNP
rs74192363	176	dbSNP
rs763521613	176	dbSNP
rs896528013	177	dbSNP
rs1196982246	184	dbSNP
rs1272077211	192	dbSNP
rs201500931	192	dbSNP
rs369117648	192	dbSNP
rs1231073842	194	dbSNP
rs1306175243	195	dbSNP
rs1357042067	197	dbSNP
rs28441279	198	dbSNP
rs73021542	202	dbSNP
rs1333855887	203	dbSNP
rs970281751	210	dbSNP
rs759962202	213	dbSNP
rs1027500846	217	dbSNP
rs1341596610	221	dbSNP
rs951723201	221	dbSNP
rs983040073	223	dbSNP
rs907530944	224	dbSNP
rs1199448701	232	dbSNP
rs73021543	233	dbSNP
rs968663529	248	dbSNP
rs1211588342	249	dbSNP
rs1235049911	249	dbSNP
rs114061406	257	dbSNP
rs1251825184	258	dbSNP
rs1211319979	264	dbSNP
rs1336438268	265	dbSNP
rs73021544	271	dbSNP
rs959735318	272	dbSNP
rs577468539	276	dbSNP
rs1348279072	279	dbSNP
rs1281138237	280	dbSNP
rs186952347	283	dbSNP
rs917987626	286	dbSNP
rs1044012417	289	dbSNP
rs970912857	291	dbSNP
rs904069350	294	dbSNP
rs1443237897	303	dbSNP
rs1395432512	307	dbSNP
rs764955272	308	dbSNP
rs1399256790	313	dbSNP
rs1052484064	316	dbSNP
rs1410621894	320	dbSNP
rs73021545	332	dbSNP
rs369562752	333	dbSNP
rs1393837339	336	dbSNP
rs1393271473	341	dbSNP
rs1013700037	343	dbSNP
rs1045536354	348	dbSNP
rs1246857274	357	dbSNP
rs905722145	368	dbSNP
rs942460544	369	dbSNP
rs1240649951	372	dbSNP
rs1040803468	374	dbSNP
rs1301187664	376	dbSNP
rs1001291208	377	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.