ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ugcAAAUUUA--AAG--U-GGGGAAGa 5' ||| | ||| | ||||||| Target 5' tctTTTCCTTCCTTCCTACCCCCTTCt 3'	423 - 449	143.00	-13.40
2	miRNA 3' ugcaaaUUUAAAGUGGGGAAGa 5' :| | || :|||||| Target 5' ctggggGACTCTCCTCCCTTCt 3'	339 - 360	136.00	-14.20
3	miRNA 3' ugcAAAU-UUAAAGUGGGGAAga 5' |||| ||||: ||:||| Target 5' tatTTTATTATTTT-CCTCTTgt 3'	754 - 775	126.00	-7.20

Mutant ID	Mutant Position	Mutant Source
1183224	109	ClinVar
638505	129	ClinVar
810608	130	ClinVar
134590	132	ClinVar
444806	198	ClinVar
1207328	361	ClinVar
1181456	491	ClinVar

Mutant ID	Mutant Position	Mutant Source
rs1208494015	4	dbSNP
rs1262967902	6	dbSNP
rs1449219860	9	dbSNP
rs1197454076	11	dbSNP
rs782749834	12	dbSNP
rs1377105835	18	dbSNP
rs782008479	25	dbSNP
rs1480832525	35	dbSNP
rs782318525	40	dbSNP
rs1178792251	41	dbSNP
rs1410074140	41	dbSNP
rs782183274	42	dbSNP
rs781971436	43	dbSNP
rs1288088245	62	dbSNP
rs1385476778	76	dbSNP
rs1383850206	83	dbSNP
rs782341771	84	dbSNP
rs1321551882	85	dbSNP
rs372057894	86	dbSNP
rs782381464	90	dbSNP
rs782647013	90	dbSNP
rs782275907	94	dbSNP
rs782215470	95	dbSNP
rs782444646	97	dbSNP
rs781849827	99	dbSNP
rs782360019	101	dbSNP
rs782003345	102	dbSNP
rs782372607	103	dbSNP
rs146836963	106	dbSNP
rs150558161	106	dbSNP
rs868923717	106	dbSNP
rs782736952	107	dbSNP
rs1201690300	109	dbSNP
rs1249821741	109	dbSNP
rs782563897	109	dbSNP
rs1472380143	114	dbSNP
rs1160198376	116	dbSNP
rs782524525	123	dbSNP
rs781897787	124	dbSNP
rs1161346740	125	dbSNP
rs782201442	130	dbSNP
rs868982577	131	dbSNP
rs781910906	132	dbSNP
rs1348155280	133	dbSNP
rs904938578	137	dbSNP
rs782737610	155	dbSNP
rs782082388	158	dbSNP
rs782001639	159	dbSNP
rs1307323422	171	dbSNP
rs782378981	179	dbSNP
rs782299839	191	dbSNP
rs782016647	195	dbSNP
rs1243109221	196	dbSNP
rs782326659	201	dbSNP
rs1024754603	205	dbSNP
rs1291607611	213	dbSNP
rs1013651789	217	dbSNP
rs1360809304	231	dbSNP
rs897509559	241	dbSNP
rs35502347	249	dbSNP
rs1225443658	263	dbSNP
rs1272569275	263	dbSNP
rs1480373800	272	dbSNP
rs782662227	284	dbSNP
rs1177977342	306	dbSNP
rs1252961394	313	dbSNP
rs1420617824	319	dbSNP
rs1185154794	341	dbSNP
rs1384440594	343	dbSNP
rs782566471	344	dbSNP
rs1425214097	347	dbSNP
rs1004477853	351	dbSNP
rs1368133501	354	dbSNP
rs888578279	355	dbSNP
rs1461063778	356	dbSNP
rs1048589544	359	dbSNP
rs139342680	361	dbSNP
rs1394592970	367	dbSNP
rs1314782205	368	dbSNP
rs1384083489	376	dbSNP
rs782663215	380	dbSNP
rs1306945023	384	dbSNP
rs1205352594	388	dbSNP
rs1253227928	395	dbSNP
rs1482389436	402	dbSNP
rs1203346555	405	dbSNP
rs1242267305	411	dbSNP
rs1445067006	413	dbSNP
rs1185290515	414	dbSNP
rs1041161088	420	dbSNP
rs1417334533	422	dbSNP
rs1427338289	423	dbSNP
rs1170550567	426	dbSNP
rs782478973	440	dbSNP
rs944015296	441	dbSNP
rs1407275904	444	dbSNP
rs1454940183	451	dbSNP
rs1319087610	459	dbSNP
rs913917275	463	dbSNP
rs1215726454	465	dbSNP
rs1310556837	466	dbSNP
rs17002644	491	dbSNP
rs1461570589	502	dbSNP
rs782710645	522	dbSNP
rs1205337016	528	dbSNP
rs1240226890	536	dbSNP
rs957953142	538	dbSNP
rs925243360	546	dbSNP
rs1420991795	562	dbSNP
rs1479200463	566	dbSNP
rs1175207299	574	dbSNP
rs980565560	576	dbSNP
rs969273014	580	dbSNP
rs1471124734	584	dbSNP
rs1316658496	592	dbSNP
rs1345035147	597	dbSNP
rs1432068396	599	dbSNP
rs782530738	601	dbSNP
rs781899618	608	dbSNP
rs1404443055	612	dbSNP
rs1284085658	624	dbSNP
rs1325943512	625	dbSNP
rs1226856322	628	dbSNP
rs782437632	628	dbSNP
rs782751699	633	dbSNP
rs183756075	654	dbSNP
rs1025121222	657	dbSNP
rs1316164737	664	dbSNP
rs1216384789	677	dbSNP
rs1243801658	695	dbSNP
rs1489617105	700	dbSNP
rs1198076814	701	dbSNP
rs1156483593	711	dbSNP
rs1269086488	711	dbSNP
rs1481208384	711	dbSNP
rs781950484	711	dbSNP
rs1401671544	716	dbSNP
rs1013348840	717	dbSNP
rs1161862033	738	dbSNP
rs1364255532	754	dbSNP
rs1424033559	763	dbSNP
rs1303824459	774	dbSNP
rs961979745	792	dbSNP
rs868979258	796	dbSNP
rs1279243678	802	dbSNP
rs1372284505	804	dbSNP
rs1224163651	805	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.