ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gaccCCACCCCUGUCUCCGAa 5' | ||| ||||||||| Target 5' agctGCTGGCCACAGAGGCTc 3'	49 - 69	161.00	-19.90
2	miRNA 3' gaccccaccccUGUCUCCGAa 5' :|||||||| Target 5' ggaacagtcctGCAGAGGCTa 3'	216 - 236	146.00	-14.10
3	miRNA 3' gaccccacCCCU-GUCUC-CGAa 5' |||| | ||| ||| Target 5' gagcccaaGGGACCTGAGTGCTg 3'	183 - 205	93.00	-13.20

Mutant ID	Mutant Position	Mutant Source
COSN31499489	12	COSMIC
COSN30454919	17	COSMIC
COSN30500853	17	COSMIC
COSN30129092	21	COSMIC
COSN27000970	25	COSMIC
COSN30526035	27	COSMIC
COSN30492175	32	COSMIC
COSN31504314	36	COSMIC
COSN27000971	58	COSMIC
COSN31557733	65	COSMIC
COSN30457559	69	COSMIC
COSN30137791	73	COSMIC
COSN28747916	78	COSMIC
COSN31571840	90	COSMIC
COSN31509693	95	COSMIC
COSN28830353	104	COSMIC
COSN30451376	120	COSMIC
COSN30542387	122	COSMIC
COSN30479017	141	COSMIC
COSN30193401	149	COSMIC
COSN20123320	167	COSMIC
COSN30121295	186	COSMIC
COSN31588379	205	COSMIC
COSN30540930	216	COSMIC
COSN31590581	245	COSMIC
COSN30541347	289	COSMIC
COSN31609596	307	COSMIC
COSN8281628	433	COSMIC
COSN24396901	455	COSMIC
COSN31550633	490	COSMIC
COSN5828162	495	COSMIC
COSN31609294	563	COSMIC
COSN25590432	610	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs747863979	7	dbSNP
rs1298516473	12	dbSNP
rs1300133238	12	dbSNP
rs1436139884	22	dbSNP
rs1387968382	25	dbSNP
rs776253410	30	dbSNP
rs376079766	33	dbSNP
rs1425858042	38	dbSNP
rs779395438	39	dbSNP
rs376549962	42	dbSNP
rs749702990	46	dbSNP
rs778284381	50	dbSNP
rs112584972	53	dbSNP
rs1410088743	62	dbSNP
rs1301920609	78	dbSNP
rs866494169	84	dbSNP
rs892645807	91	dbSNP
rs978801114	92	dbSNP
rs569770350	94	dbSNP
rs1344533282	98	dbSNP
rs1206141170	101	dbSNP
rs1291056870	105	dbSNP
rs921238809	106	dbSNP
rs145712356	109	dbSNP
rs141849007	110	dbSNP
rs796865645	111	dbSNP
rs955104186	112	dbSNP
rs559535543	124	dbSNP
rs548047676	126	dbSNP
rs915541647	129	dbSNP
rs983060073	132	dbSNP
rs1388220735	133	dbSNP
rs532729986	141	dbSNP
rs951239469	147	dbSNP
rs1026765473	149	dbSNP
rs1343256566	151	dbSNP
rs938606607	154	dbSNP
rs919129312	156	dbSNP
rs151315992	161	dbSNP
rs774216381	163	dbSNP
rs1435048674	166	dbSNP
rs1023278981	172	dbSNP
rs1457724599	181	dbSNP
rs1367809640	183	dbSNP
rs1229678970	193	dbSNP
rs963456883	195	dbSNP
rs1354559260	203	dbSNP
rs909311826	206	dbSNP
rs775711482	211	dbSNP
rs1290720014	219	dbSNP
rs1414045193	222	dbSNP
rs1013185033	224	dbSNP
rs1480119839	225	dbSNP
rs544002837	234	dbSNP
rs986209284	239	dbSNP
rs116183548	244	dbSNP
rs1057378145	245	dbSNP
rs1030297513	255	dbSNP
rs1241913992	260	dbSNP
rs867315005	264	dbSNP
rs1163288047	270	dbSNP
rs1216897523	273	dbSNP
rs1383619996	274	dbSNP
rs748790152	284	dbSNP
rs976774505	290	dbSNP
rs956924839	292	dbSNP
rs185253171	294	dbSNP
rs78546575	303	dbSNP
rs1028523625	305	dbSNP
rs1223422329	307	dbSNP
rs1043138340	309	dbSNP
rs1338884247	315	dbSNP
rs947444299	323	dbSNP
rs1247268580	331	dbSNP
rs903999321	332	dbSNP
rs1226725322	337	dbSNP
rs1269914233	339	dbSNP
rs1021798509	348	dbSNP
rs1199611526	353	dbSNP
rs1301320232	361	dbSNP
rs1253850277	370	dbSNP
rs921271677	390	dbSNP
rs182120262	391	dbSNP
rs745517532	392	dbSNP
rs757979220	401	dbSNP
rs552721220	407	dbSNP
rs534901523	420	dbSNP
rs577027885	425	dbSNP
rs894168827	438	dbSNP
rs780933106	442	dbSNP
rs1172292428	450	dbSNP
rs938620211	459	dbSNP
rs1390766248	467	dbSNP
rs1398219269	468	dbSNP
rs142513222	469	dbSNP
rs1335102217	474	dbSNP
rs951313372	481	dbSNP
rs1469227978	489	dbSNP
rs1408059715	490	dbSNP
rs1290322062	495	dbSNP
rs897731027	497	dbSNP
rs1026796228	499	dbSNP
rs1178262045	505	dbSNP
rs1232442643	514	dbSNP
rs1253962174	516	dbSNP
rs1346847058	521	dbSNP
rs1426990100	535	dbSNP
rs1374733375	538	dbSNP
rs973834632	539	dbSNP
rs1481897525	542	dbSNP
rs1183663149	543	dbSNP
rs1418987207	544	dbSNP
rs536811435	551	dbSNP
rs969095272	553	dbSNP
rs1420142028	555	dbSNP
rs1023309993	569	dbSNP
rs1175957966	570	dbSNP
rs1044392	573	dbSNP
rs753101076	574	dbSNP
rs1173574151	575	dbSNP
rs1370130860	586	dbSNP
rs986627298	592	dbSNP
rs1036375456	603	dbSNP
rs923290758	606	dbSNP
rs976098003	607	dbSNP
rs956662652	608	dbSNP
rs748031511	610	dbSNP
rs1332801647	616	dbSNP
rs548273386	619	dbSNP
rs536377854	620	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.