ID	Duplex structure	Position	Score	MFE
1	miRNA 3' agAC-UAG--UCCGUUU----CAACGUCc 5' || ||| | |||| ||| :|| Target 5' ctTGTATCCAAAACAAATCAGGTTTTAGa 3'	221 - 249	104.00	-7.76
2	miRNA 3' agACUAGUCCGUU-----UCAACG-UCc 5' | || | |||| | |||| || Target 5' ctTTAT-AAGCAATTTATATTTGCTAGa 3'	95 - 121	91.00	-5.60
3	miRNA 3' agacuaguccguUUCAA--CGUCc 5' |:||| |:|| Target 5' gcactctttcttAGGTTCGGTAGa 3'	271 - 294	90.00	-6.50

Mutant ID	Mutant Position	Mutant Source
COSN18316042	368	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs4328177	1	dbSNP
rs544634704	12	dbSNP
rs1250202499	13	dbSNP
rs1445496750	15	dbSNP
rs1192210412	19	dbSNP
rs1237782440	24	dbSNP
rs755850829	32	dbSNP
rs1473750326	33	dbSNP
rs747760157	34	dbSNP
rs1248261789	36	dbSNP
rs780674156	42	dbSNP
rs754561961	44	dbSNP
rs1356480021	45	dbSNP
rs1417038827	62	dbSNP
rs1457357342	63	dbSNP
rs1032807847	64	dbSNP
rs1279164304	77	dbSNP
rs1045826771	88	dbSNP
rs573034696	110	dbSNP
rs996021189	111	dbSNP
rs894203209	117	dbSNP
rs1208171940	118	dbSNP
rs1341402804	120	dbSNP
rs1416973663	121	dbSNP
rs568338846	122	dbSNP
rs1303055338	123	dbSNP
rs1262504454	126	dbSNP
rs564983570	131	dbSNP
rs565583109	134	dbSNP
rs944666546	140	dbSNP
rs1185502071	146	dbSNP
rs141459910	147	dbSNP
rs1472838993	154	dbSNP
rs1047408234	156	dbSNP
rs573426045	160	dbSNP
rs554861864	172	dbSNP
rs1172838801	177	dbSNP
rs113120563	178	dbSNP
rs945947565	181	dbSNP
rs112582978	182	dbSNP
rs920352936	198	dbSNP
rs990558456	200	dbSNP
rs1309272481	215	dbSNP
rs922601226	219	dbSNP
rs575232307	222	dbSNP
rs1275715588	225	dbSNP
rs977145259	228	dbSNP
rs964100936	234	dbSNP
rs1279268708	240	dbSNP
rs928175853	242	dbSNP
rs911296231	252	dbSNP
rs1248283915	254	dbSNP
rs368782604	256	dbSNP
rs538269295	273	dbSNP
rs1198932960	276	dbSNP
rs966993165	279	dbSNP
rs1248471133	281	dbSNP
rs952850843	284	dbSNP
rs367610062	285	dbSNP
rs1025772131	288	dbSNP
rs1259137218	289	dbSNP
rs111458191	304	dbSNP
rs1220900932	313	dbSNP
rs146058891	324	dbSNP
rs1394944200	327	dbSNP
rs1390952211	338	dbSNP
rs970965863	340	dbSNP
rs1324279487	349	dbSNP
rs1301268272	356	dbSNP
rs1373485910	363	dbSNP
rs1232304593	370	dbSNP
rs989513646	379	dbSNP
rs554885728	380	dbSNP
rs1025252510	386	dbSNP
rs1014317059	387	dbSNP
rs1229307714	388	dbSNP
rs1249602347	389	dbSNP
rs1367715852	394	dbSNP
rs1480263633	399	dbSNP
rs1294729227	400	dbSNP
rs751031179	418	dbSNP
rs766348688	419	dbSNP
rs766409773	421	dbSNP
rs758276867	425	dbSNP
rs1369550330	429	dbSNP
rs546496612	432	dbSNP
rs1209922067	433	dbSNP
rs202166634	434	dbSNP
rs764978526	437	dbSNP
rs753929231	444	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.