ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cuuUCU-GUCAU--CUAACAUAUc 5' ||| :| || |||| |||| Target 5' tttAGATTAATACTGATTATATAg 3'	125 - 148	128.00	-6.60
2	miRNA 3' cuUUCUGUC-----AUCU--AACAUAuc 5' || :||| |||| ||||:| Target 5' ggAAAGCAGATGCTTAGATTTTGTGTca 3'	62 - 89	121.00	-9.80
3	miRNA 3' cuuuCUGUCAUCUAACAUauc 5' | || | |:||||| Target 5' ttctGTCACT-GGTTGTAaga 3'	922 - 941	112.00	-6.60

Mutant ID	Mutant Position	Mutant Source
COSN30111388	18	COSMIC
COSN30123828	27	COSMIC
COSN30165811	31	COSMIC
COSN30529174	101	COSMIC
COSN31510595	128	COSMIC
COSN30177399	146	COSMIC
COSN31543655	150	COSMIC
COSN30536628	164	COSMIC
COSN31585455	204	COSMIC
COSN16958302	462	COSMIC
COSN20966474	593	COSMIC
COSN7694032	613	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs374807873	3	dbSNP
rs776224862	4	dbSNP
rs770368270	18	dbSNP
rs760173897	24	dbSNP
rs774724138	26	dbSNP
rs199529936	29	dbSNP
rs1474724429	30	dbSNP
rs1188278282	31	dbSNP
rs1355756168	35	dbSNP
rs749724789	38	dbSNP
rs1451549197	44	dbSNP
rs750765344	54	dbSNP
rs772233371	70	dbSNP
rs545991228	84	dbSNP
rs1405971415	90	dbSNP
rs575461000	97	dbSNP
rs1237108405	107	dbSNP
rs561855961	108	dbSNP
rs1013931820	110	dbSNP
rs1041681459	115	dbSNP
rs542021908	129	dbSNP
rs1243312517	137	dbSNP
rs1313833967	143	dbSNP
rs189949323	148	dbSNP
rs1211056449	153	dbSNP
rs74367049	154	dbSNP
rs1445741402	158	dbSNP
rs1015173672	164	dbSNP
rs1249036376	165	dbSNP
rs1182149913	173	dbSNP
rs1451651787	173	dbSNP
rs1004554627	174	dbSNP
rs1473268053	175	dbSNP
rs1165045757	179	dbSNP
rs931960077	185	dbSNP
rs1439730411	188	dbSNP
rs1456627640	204	dbSNP
rs779340888	213	dbSNP
rs976184920	216	dbSNP
rs1450114002	226	dbSNP
rs1290265001	248	dbSNP
rs887560777	248	dbSNP
rs1027275923	253	dbSNP
rs966640268	257	dbSNP
rs1451070562	259	dbSNP
rs1344069708	260	dbSNP
rs908031832	260	dbSNP
rs1269260067	261	dbSNP
rs1466658839	264	dbSNP
rs566294499	265	dbSNP
rs900071496	266	dbSNP
rs1443193510	274	dbSNP
rs983558874	281	dbSNP
rs1039927309	289	dbSNP
rs952306966	298	dbSNP
rs553095625	299	dbSNP
rs1414030112	300	dbSNP
rs974185058	303	dbSNP
rs1468193912	310	dbSNP
rs1449837599	311	dbSNP
rs1171745771	321	dbSNP
rs1264194650	331	dbSNP
rs768984800	334	dbSNP
rs958391704	340	dbSNP
rs149457606	343	dbSNP
rs1397400730	348	dbSNP
rs1326373919	356	dbSNP
rs1349514748	362	dbSNP
rs115247701	368	dbSNP
rs1002610607	369	dbSNP
rs906930773	377	dbSNP
rs1020144820	384	dbSNP
rs1052614279	386	dbSNP
rs1231769608	408	dbSNP
rs1255757066	410	dbSNP
rs1256627795	426	dbSNP
rs1202547776	427	dbSNP
rs7773304	439	dbSNP
rs925381570	440	dbSNP
rs1182937509	458	dbSNP
rs781007895	458	dbSNP
rs979413024	459	dbSNP
rs1198143764	461	dbSNP
rs1054369616	462	dbSNP
rs1467340006	466	dbSNP
rs1165965839	467	dbSNP
rs536666336	469	dbSNP
rs916587295	471	dbSNP
rs1309293333	474	dbSNP
rs932009535	478	dbSNP
rs137953320	479	dbSNP
rs1040881781	481	dbSNP
rs1332192580	494	dbSNP
rs944827092	502	dbSNP
rs1328553975	509	dbSNP
rs907931927	511	dbSNP
rs991361408	517	dbSNP
rs554875035	519	dbSNP
rs1341681043	529	dbSNP
rs1297336408	535	dbSNP
rs1276843562	537	dbSNP
rs1380139280	542	dbSNP
rs1487582310	545	dbSNP
rs1209337236	547	dbSNP
rs548077454	548	dbSNP
rs879517603	549	dbSNP
rs1312890330	552	dbSNP
rs1014701584	565	dbSNP
rs983586749	566	dbSNP
rs1449686445	569	dbSNP
rs1170164340	572	dbSNP
rs1363336030	575	dbSNP
rs1456916573	578	dbSNP
rs1373626539	579	dbSNP
rs951781704	581	dbSNP
rs930810217	588	dbSNP
rs995895766	591	dbSNP
rs1364994320	598	dbSNP
rs1374549700	606	dbSNP
rs1191348398	607	dbSNP
rs115878264	612	dbSNP
rs62442791	613	dbSNP
rs1019031323	616	dbSNP
rs1258335809	622	dbSNP
rs577125373	622	dbSNP
rs1206811440	624	dbSNP
rs1008447941	626	dbSNP
rs991075956	626	dbSNP
rs1190079161	643	dbSNP
rs1190288348	644	dbSNP
rs1382952679	645	dbSNP
rs958640935	649	dbSNP
rs1164826033	656	dbSNP
rs1369743253	663	dbSNP
rs935512783	675	dbSNP
rs1319209746	679	dbSNP
rs1388549212	689	dbSNP
rs1436216912	691	dbSNP
rs746915085	699	dbSNP
rs904033557	704	dbSNP
rs1228015292	707	dbSNP
rs1034038495	726	dbSNP
rs565977432	729	dbSNP
rs1043807554	740	dbSNP
rs948138147	743	dbSNP
rs186721596	744	dbSNP
rs1329799334	748	dbSNP
rs1224804769	757	dbSNP
rs1306489768	762	dbSNP
rs992600591	764	dbSNP
rs557224671	765	dbSNP
rs939280230	773	dbSNP
rs1182141631	777	dbSNP
rs1257652878	780	dbSNP
rs1471284210	784	dbSNP
rs1157785112	792	dbSNP
rs1020004405	793	dbSNP
rs907821803	811	dbSNP
rs1161801868	825	dbSNP
rs1458268578	825	dbSNP
rs1416148902	826	dbSNP
rs1010110634	831	dbSNP
rs1447568052	833	dbSNP
rs1303577762	849	dbSNP
rs1381637277	853	dbSNP
rs1408022365	861	dbSNP
rs1287841861	862	dbSNP
rs1350997811	868	dbSNP
rs1230203867	880	dbSNP
rs1276224496	888	dbSNP
rs983236727	892	dbSNP
rs951853453	897	dbSNP
rs892965052	901	dbSNP
rs1257331083	903	dbSNP
rs1312237591	905	dbSNP
rs1027463434	908	dbSNP
rs1251974955	912	dbSNP
rs1473199066	930	dbSNP
rs974482030	940	dbSNP
rs1033371563	942	dbSNP
rs1423692465	946	dbSNP
rs996089644	955	dbSNP
rs900392792	956	dbSNP
rs1040387044	958	dbSNP
rs1177277461	959	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.