ID	Duplex structure	Position	Score	MFE
1	miRNA 3' acaUCACGUUGAUACGUUUUGa 5' || | || |: ||||||: Target 5' cagAGGGGAA-TG-GCAAAATg 3'	337 - 356	133.00	-6.50
2	miRNA 3' acAUCACGUUGAUACGUUUUGa 5' ||||| ||: | ||||| Target 5' ggTAGTG---CTG-GAAAAACg 3'	244 - 261	124.00	-10.50
3	miRNA 3' acaucACGUU---GAUACGUUUUga 5' ||||| | | |||||: Target 5' taacaTGCAAGGGCCAAGCAAAGgc 3'	290 - 314	121.00	-9.00

Mutant ID	Mutant Position	Mutant Source
COSN30143889	9	COSMIC
COSN30499364	13	COSMIC
COSN4891702	14	COSMIC
COSN30500498	47	COSMIC
COSN9780855	71	COSMIC
COSN30530060	77	COSMIC
COSN30141239	83	COSMIC
COSN30109930	178	COSMIC
COSN31533191	365	COSMIC
COSN30172580	387	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1334180310	1	dbSNP
rs200690743	7	dbSNP
rs367648977	8	dbSNP
rs780213812	9	dbSNP
rs956044111	10	dbSNP
rs756351984	11	dbSNP
rs1258644120	12	dbSNP
rs1485326023	13	dbSNP
rs201315242	13	dbSNP
rs370640530	14	dbSNP
rs757348758	16	dbSNP
rs1192239476	20	dbSNP
rs752620770	23	dbSNP
rs1181893528	29	dbSNP
rs1394560687	30	dbSNP
rs368396447	31	dbSNP
rs759364487	32	dbSNP
rs753703708	34	dbSNP
rs374487579	40	dbSNP
rs760370358	41	dbSNP
rs750493218	43	dbSNP
rs1255056732	50	dbSNP
rs771601121	51	dbSNP
rs761245551	57	dbSNP
rs935325810	59	dbSNP
rs200689983	62	dbSNP
rs11552399	67	dbSNP
rs1376925176	68	dbSNP
rs551404853	73	dbSNP
rs1302507945	74	dbSNP
rs749605973	78	dbSNP
rs1351231429	79	dbSNP
rs780374959	85	dbSNP
rs879744040	93	dbSNP
rs1406311719	95	dbSNP
rs770185828	101	dbSNP
rs1315996943	125	dbSNP
rs1354465779	127	dbSNP
rs757912725	135	dbSNP
rs1166189387	144	dbSNP
rs1407420015	159	dbSNP
rs532525594	167	dbSNP
rs112924363	173	dbSNP
rs371499570	175	dbSNP
rs1274211037	177	dbSNP
rs888257167	183	dbSNP
rs1252988139	193	dbSNP
rs1412137466	196	dbSNP
rs1179067857	202	dbSNP
rs1405837983	205	dbSNP
rs1164439527	211	dbSNP
rs1418746840	212	dbSNP
rs1165857400	213	dbSNP
rs190926940	220	dbSNP
rs1459744176	233	dbSNP
rs1473058216	234	dbSNP
rs1048807645	237	dbSNP
rs1400987651	240	dbSNP
rs539712040	245	dbSNP
rs7754065	253	dbSNP
rs916375408	257	dbSNP
rs185862325	260	dbSNP
rs983967074	261	dbSNP
rs372915880	267	dbSNP
rs1294817220	277	dbSNP
rs182519922	278	dbSNP
rs1040723740	280	dbSNP
rs1272911698	282	dbSNP
rs1025378703	284	dbSNP
rs1207184985	285	dbSNP
rs549703792	294	dbSNP
rs1801823	297	dbSNP
rs1188727149	299	dbSNP
rs1259951116	302	dbSNP
rs1019221287	314	dbSNP
rs1228574643	315	dbSNP
rs1330792267	318	dbSNP
rs1411597531	320	dbSNP
rs1168164305	322	dbSNP
rs1009121889	331	dbSNP
rs889291758	337	dbSNP
rs1414937611	344	dbSNP
rs1285838523	355	dbSNP
rs1227237405	357	dbSNP
rs1029617704	365	dbSNP
rs775682699	366	dbSNP
rs1455849103	370	dbSNP
rs1413734931	373	dbSNP
rs1294607146	381	dbSNP
rs1293093766	382	dbSNP
rs189428717	388	dbSNP
rs1340409974	389	dbSNP
rs1439266666	396	dbSNP
rs1216840162	397	dbSNP
rs1271571895	401	dbSNP
rs1211754522	405	dbSNP
rs1307418582	405	dbSNP
rs1348458191	408	dbSNP
rs772078928	417	dbSNP
rs914708862	419	dbSNP
rs989476737	446	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.