ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gaGGUAAAAGGUGGU-CUCGACGu 5' :||| || : :|| ||||||| Target 5' agTCATGTTATCTCATGAGCTGCt 3'	437 - 460	161.00	-16.30
2	miRNA 3' gagGUAAA------AGGUGGUCUCGACGu 5' ||||| |: ::| | ||||| Target 5' ttgCATTTGGCTAATTGGTCTGTGCTGCa 3'	177 - 205	122.00	-13.30
3	miRNA 3' gaGGUAAAAGGUGGUCUCGACGu 5' | ||| ||||: || :||| Target 5' acCAATTAACCACT-GAATTGCt 3'	23 - 44	116.00	-13.90

Mutant ID	Mutant Position	Mutant Source
COSN23914966	4	COSMIC
COSN30148097	14	COSMIC
COSN30492909	22	COSMIC
COSN31550984	25	COSMIC
COSN31592959	35	COSMIC
COSN30491737	37	COSMIC
COSN26996809	43	COSMIC
COSN30103455	69	COSMIC
COSN30453943	71	COSMIC
COSN30162251	101	COSMIC
COSN30540272	113	COSMIC
COSN30540298	114	COSMIC
COSN23949752	118	COSMIC
COSN31604634	163	COSMIC
COSN19660625	241	COSMIC
COSN30160689	268	COSMIC
COSN31588445	278	COSMIC
COSN23341424	479	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs778890477	4	dbSNP
rs771086831	6	dbSNP
rs1325628646	9	dbSNP
rs1479332958	9	dbSNP
rs149732177	17	dbSNP
rs778184279	18	dbSNP
rs764336389	19	dbSNP
rs1335521521	24	dbSNP
rs1469826405	25	dbSNP
rs1394659357	27	dbSNP
rs1464777346	32	dbSNP
rs200415110	33	dbSNP
rs1179134267	44	dbSNP
rs1402844811	44	dbSNP
rs935950622	45	dbSNP
rs1372836702	46	dbSNP
rs1393957269	52	dbSNP
rs11542831	55	dbSNP
rs1377638691	61	dbSNP
rs1230589590	62	dbSNP
rs1293978814	68	dbSNP
rs1317414629	79	dbSNP
rs113051637	83	dbSNP
rs1059613	87	dbSNP
rs1455607849	92	dbSNP
rs965661536	104	dbSNP
rs1018663790	118	dbSNP
rs150964319	121	dbSNP
rs1190069236	122	dbSNP
rs963408191	123	dbSNP
rs983636228	124	dbSNP
rs1017195825	130	dbSNP
rs985671716	144	dbSNP
rs759110855	152	dbSNP
rs1194912120	156	dbSNP
rs559410717	159	dbSNP
rs1410824279	161	dbSNP
rs549167506	173	dbSNP
rs1454459020	174	dbSNP
rs998781056	178	dbSNP
rs529086951	189	dbSNP
rs1314077937	191	dbSNP
rs770923604	201	dbSNP
rs995011060	205	dbSNP
rs1280264157	207	dbSNP
rs1340343928	212	dbSNP
rs895400731	213	dbSNP
rs1207915749	217	dbSNP
rs868489633	219	dbSNP
rs1010900879	223	dbSNP
rs1033905764	230	dbSNP
rs1003556703	233	dbSNP
rs4770	242	dbSNP
rs1487635275	245	dbSNP
rs1190891242	248	dbSNP
rs1243403280	255	dbSNP
rs1171677125	258	dbSNP
rs1214948196	258	dbSNP
rs1055054183	273	dbSNP
rs543717605	279	dbSNP
rs1042372157	280	dbSNP
rs1436996658	282	dbSNP
rs773553966	283	dbSNP
rs906476163	288	dbSNP
rs1046210209	297	dbSNP
rs893881884	304	dbSNP
rs1054703186	306	dbSNP
rs1435991144	316	dbSNP
rs929159085	319	dbSNP
rs935810679	320	dbSNP
rs758946184	323	dbSNP
rs1370535306	331	dbSNP
rs577987510	334	dbSNP
rs921871229	344	dbSNP
rs564098990	346	dbSNP
rs1280881490	353	dbSNP
rs919004752	357	dbSNP
rs182952959	358	dbSNP
rs371579719	366	dbSNP
rs572214633	375	dbSNP
rs1363064602	377	dbSNP
rs1288834775	379	dbSNP
rs1447125315	383	dbSNP
rs1197662685	389	dbSNP
rs1224647295	391	dbSNP
rs944613497	392	dbSNP
rs1377739663	395	dbSNP
rs941763543	402	dbSNP
rs1174984544	403	dbSNP
rs1380140663	407	dbSNP
rs910185837	414	dbSNP
rs1418407694	419	dbSNP
rs555450972	432	dbSNP
rs1160846817	434	dbSNP
rs1434798178	440	dbSNP
rs1404868812	443	dbSNP
rs985807476	450	dbSNP
rs954256633	463	dbSNP
rs1373480480	466	dbSNP
rs1441987490	473	dbSNP
rs554509500	479	dbSNP
rs1375927596	483	dbSNP
rs1236762549	486	dbSNP
rs976935754	488	dbSNP
rs1350716297	490	dbSNP
rs1185665809	491	dbSNP
rs1287233195	491	dbSNP
rs950558261	492	dbSNP
rs967316326	504	dbSNP
rs1282296132	505	dbSNP
rs1237927887	506	dbSNP
rs1021123805	509	dbSNP
rs1184543897	513	dbSNP
rs748438511	518	dbSNP
rs1472474385	520	dbSNP
rs958126956	527	dbSNP
rs1347219905	553	dbSNP
rs1033615671	556	dbSNP
rs1033833799	559	dbSNP
rs1399212182	560	dbSNP
rs1002184074	567	dbSNP
rs1330735036	582	dbSNP
rs143972040	590	dbSNP
rs1295327989	597	dbSNP
rs906446796	598	dbSNP
rs17848506	606	dbSNP
rs1233502291	614	dbSNP
rs1257061227	615	dbSNP
rs1456666793	626	dbSNP
rs993341509	631	dbSNP
rs1251757239	636	dbSNP
rs1442341687	638	dbSNP
rs1186096781	641	dbSNP
rs893884873	644	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.