Inhibition of Both HIV-1 Reverse Transcription and Gene Expression by
a Cyclic Peptide that Binds the Tat-Transactivating Response Element (TAR)
RNA
The RNA response element TAR plays a critical role in HIV replication by
providing a binding site for the recruitment of the viral transactivator protein
Tat. Using a structure-guided approach, we have developed a series of
conformationally-constrained cyclic peptides that act as structural mimics of
the Tat RNA binding region and block Tat-TAR interactions at nanomolar
concentrations in vitro. Here we show that these compounds
block Tat-dependent transcription in cell-free systems and in cell-based
reporter assays. The compounds are also cell permeable, have low toxicity, and
inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and
CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and
CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic
L50 exhibited an IC50 ∼250 nM. Surprisingly, inhibition of
LTR-driven HIV-1 transcription could not account for the full antiviral
activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic
inhibition curve with the first phase occurring after HIV-1 entry into the host
cell and during the initiation of HIV-1 reverse transcription. The second phase
coincides with inhibition of HIV-1 transcription. Reconstituted reverse
transcription assays confirm that HIV-1 (−) strand strong stop DNA
synthesis is blocked by L50-TAR RNA interactions in-vitro.
These findings are consistent with genetic evidence that TAR plays critical
roles both during reverse transcription and during HIV gene expression. Our
results suggest that antiviral drugs targeting TAR RNA might be highly effective
due to a dual inhibitory mechanism.
Vyšlo v časopise:
Inhibition of Both HIV-1 Reverse Transcription and Gene Expression by
a Cyclic Peptide that Binds the Tat-Transactivating Response Element (TAR)
RNA. PLoS Pathog 7(5): e32767. doi:10.1371/journal.ppat.1002038
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.ppat.1002038
Souhrn
The RNA response element TAR plays a critical role in HIV replication by
providing a binding site for the recruitment of the viral transactivator protein
Tat. Using a structure-guided approach, we have developed a series of
conformationally-constrained cyclic peptides that act as structural mimics of
the Tat RNA binding region and block Tat-TAR interactions at nanomolar
concentrations in vitro. Here we show that these compounds
block Tat-dependent transcription in cell-free systems and in cell-based
reporter assays. The compounds are also cell permeable, have low toxicity, and
inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and
CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and
CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic
L50 exhibited an IC50 ∼250 nM. Surprisingly, inhibition of
LTR-driven HIV-1 transcription could not account for the full antiviral
activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic
inhibition curve with the first phase occurring after HIV-1 entry into the host
cell and during the initiation of HIV-1 reverse transcription. The second phase
coincides with inhibition of HIV-1 transcription. Reconstituted reverse
transcription assays confirm that HIV-1 (−) strand strong stop DNA
synthesis is blocked by L50-TAR RNA interactions in-vitro.
These findings are consistent with genetic evidence that TAR plays critical
roles both during reverse transcription and during HIV gene expression. Our
results suggest that antiviral drugs targeting TAR RNA might be highly effective
due to a dual inhibitory mechanism.
Zdroje
1. VolberdingPADeeksSG
2010
Antiretroviral therapy and management of HIV
infection.
Lancet
376
49
62
2. RichmanDDMargolisDMDelaneyMGreeneWCHazudaD
2009
The challenge of finding a cure for HIV
infection.
Science
323
1304
1307
3. Menendez-AriasL
2010
Molecular basis of human immunodeficiency virus drug resistance:
an update.
Antiviral Res
85
210
231
4. TaiwoBHicksCEronJ
2010
Unmet therapeutic needs in the new era of combination
antiretroviral therapy for HIV-1.
J Antimicrob Chemother
65
1100
1107
5. KarnJ
1999
Tackling Tat.
J Mol Biol
293
235
254
6. PeterlinBMPriceDH
2006
Controlling the elongation phase of transcription with
P-TEFb.
Mol Cell
23
297
305
7. WeiPGarberMEFangSMFischerWHJonesKA
1998
A novel CDK9-associated C-type cyclin interacts directly with
HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR
RNA.
Cell
92
451
462
8. KimYKBourgeoisCFIselCChurcherMJKarnJ
2002
Phosphorylation of the RNA polymerase II carboxyl-terminal domain
by CDK9 is directly responsible for human immunodeficiency virus type 1
Tat-activated transcriptional elongation.
Mol Cell Biol
22
4622
4637
9. CupelliLAHsuMC
1995
The human immunodeficiency virus type 1 Tat antagonist, Ro
5-3335, predominantly inhibits transcription initiation from the viral
promoter.
J Virol
69
2640
2643
10. HwangSTamilarasuNKiblerKCaoHAliA
2003
Discovery of a small molecule Tat-trans-activation-responsive RNA
antagonist that potently inhibits human immunodeficiency virus-1
replication.
J Biol Chem
278
39092
39103
11. FujinagaKIrwinDHuangYTaubeRKurosuT
2004
Dynamics of human immunodeficiency virus transcription: P-TEFb
phosphorylates RD and dissociates negative effectors from the
transactivation response element.
Mol Cell Biol
24
787
795
12. TahirovTHBabayevaNDVarzavandKCooperJJSedoreSC
2010
Crystal structure of HIV-1 Tat complexed with human
P-TEFb.
Nature
465
747
751
13. KarnJ
2011
The molecular biology of HIV latency: breaking and restoring the
Tat-dependent transcriptional circuit.
Curr Opin HIV AIDS
6
4
11
14. DavisBAfsharMVaraniGMurchieAIKarnJ
2004
Rational design of inhibitors of HIV-1 TAR RNA through the
stabilisation of electrostatic “hot spots”.
J Mol Biol
336
343
356
15. MurchieAIDavisBIselCAfsharMDrysdaleMJ
2004
Structure-based drug design targeting an inactive RNA
conformation: exploiting the flexibility of HIV-1 TAR RNA.
J Mol Biol
336
625
638
16. HamyFBrondaniVFlorsheimerAStarkWBlommersMJ
1998
A new class of HIV-1 Tat antagonist acting through Tat-TAR
inhibition.
Biochemistry
37
5086
5095
17. HamyFFelderERHeizmannGLazdinsJAboul-elaF
1997
An inhibitor of the Tat/TAR RNA interaction that effectively
suppresses HIV-1 replication.
Proc Natl Acad Sci U S A
94
3548
3553
18. O'BrienWASumner-SmithMMaoSHSadeghiSZhaoJQ
1996
Anti-human immunodeficiency virus type 1 activity of an
oligocationic compound mediated via gp120 V3 interactions.
J Virol
70
2825
2831
19. HsuMCSchuttADHollyMSliceLWShermanMI
1991
Inhibition of HIV replication in acute and chronic infections in
vitro by a Tat antagonist.
Science
254
1799
1802
20. HuqIPingYHTamilarasuNRanaTM
1999
Controlling human immunodeficiency virus type 1 gene expression
by unnatural peptides.
Biochemistry
38
5172
5177
21. LeeCWCaoHIchiyamaKRanaTM
2005
Design and synthesis of a novel peptidomimetic inhibitor of HIV-1
Tat-TAR interactions: squaryldiamide as a new potential bioisostere of
unsubstituted guanidine.
Bioorg Med Chem Lett
15
4243
4246
22. DoranzBJGrovit-FerbasKSharronMPMaoSHGoetzMB
1997
A small-molecule inhibitor directed against the chemokine
receptor CXCR4 prevents its use as an HIV-1 coreceptor.
J Exp Med
186
1395
1400
23. DaelemansDScholsDWitvrouwMPannecouqueCHatseS
2000
A second target for the peptoid Tat/transactivation response
element inhibitor CGP64222: inhibition of human immunodeficiency virus
replication by blocking CXC-chemokine receptor 4-mediated virus
entry.
Mol Pharmacol
57
116
124
24. AthanassiouZDiasRLMoehleKDobsonNVaraniG
2004
Structural mimicry of retroviral tat proteins by constrained
beta-hairpin peptidomimetics: ligands with high affinity and selectivity for
viral TAR RNA regulatory elements.
J Am Chem Soc
126
6906
6913
25. AthanassiouZPatoraKDiasRLMoehleKRobinsonJA
2007
Structure-guided peptidomimetic design leads to nanomolar
beta-hairpin inhibitors of the Tat-TAR interaction of bovine
immunodeficiency virus.
Biochemistry
46
741
751
26. DavidsonALeeperTCAthanassiouZPatora-KomisarskaKKarnJ
2009
Simultaneous recognition of HIV-1 TAR RNA bulge and loop
sequences by cyclic peptide mimics of Tat protein.
Proc Natl Acad Sci U S A
106
11931
11936
27. LeeperTCAthanassiouZDiasRLRobinsonJAVaraniG
2005
TAR RNA recognition by a cyclic peptidomimetic of Tat
protein.
Biochemistry
44
12362
12372
28. PuglisiJDChenLBlanchardSFrankelAD
1995
Solution structure of a bovine immunodeficiency virus Tat-TAR
peptide-RNA complex.
Science
270
1200
1203
29. YeXKumarRAPatelDJ
1995
Molecular recognition in the bovine immunodeficiency virus Tat
peptide-TAR RNA complex.
Chem Biol
2
827
840
30. DudleyDMWentzelJLLalondeMSVeazeyRSArtsEJ
2009
Selection of a simian-human immunodeficiency virus strain
resistant to a vaginal microbicide in macaques.
J Virol
83
5067
5076
31. WildCOasTMcDanalCBolognesiDMatthewsT
1992
A synthetic peptide inhibitor of human immunodeficiency virus
replication: correlation between solution structure and viral
inhibition.
Proc Natl Acad Sci U S A
89
10537
10541
32. RichardJPMelikovKVivesERamosCVerbeureB
2003
Cell-penetrating peptides. A reevaluation of the mechanism of
cellular uptake.
J Biol Chem
278
585
590
33. MannDAFrankelAD
1991
Endocytosis and targeting of exogenous HIV-1 Tat
protein.
EMBO J
10
1733
1739
34. ZieglerANerviPDurrenbergerMSeeligJ
2005
The cationic cell-penetrating peptide CPP(TAT) derived from the
HIV-1 protein TAT is rapidly transported into living fibroblasts: optical,
biophysical, and metabolic evidence.
Biochemistry
44
138
148
35. TakeuchiTKosugeMTadokoroASugiuraYNishiM
2006
Direct and rapid cytosolic delivery using cell-penetrating
peptides mediated by pyrenebutyrate.
ACS Chem Biol
1
299
303
36. MacaraIG
2001
Transport into and out of the nucleus.
Microbiol Mol Biol Rev
65
570
94, table
37. ScottMSBoisvertFMMcDowallMDLamondAIBartonGJ
2010
Characterization and prediction of protein nucleolar localization
sequences.
Nucleic Acids Res
38
7388
7399
38. ArienKKVanhamGArtsEJ
2007
Is HIV-1 evolving to a less virulent form in
humans?
Nat Rev Microbiol
5
141
151
39. BourgeoisCFKimYKChurcherMJWestMJKarnJ
2002
Spt5 cooperates with human immunodeficiency virus type 1 Tat by
preventing premature RNA release at terminator sequences.
Mol Cell Biol
22
1079
1093
40. GraebleMAChurcherMJLoweADGaitMJKarnJ
1993
Human immunodeficiency virus type 1 transactivator protein, tat,
stimulates transcriptional read-through of distal terminator sequences in
vitro.
Proc Natl Acad Sci U S A
90
6184
6188
41. WeberJWeberovaJCarobeneMMirzaMMartinez-PicadoJ
2006
Use of a novel assay based on intact recombinant viruses
expressing green (EGFP) or red (DsRed2) fluorescent proteins to examine the
contribution of pol and env genes to overall HIV-1 replicative
fitness.
J Virol Methods
136
102
117
42. LassenKGLobritzMABaileyJRJohnstonSNguyenS
2009
Elite suppressor-derived HIV-1 envelope glycoproteins exhibit
reduced entry efficiency and kinetics.
PLoS Pathog
5
e1000377
43. ManceboHSLeeGFlygareJTomassiniJLuuP
1997
P-TEFb kinase is required for HIV Tat transcriptional activation
in vivo and in vitro.
Genes Dev
11
2633
2644
44. WestMJLoweADKarnJ
2001
Activation of human immunodeficiency virus transcription in T
cells revisited: NF-kappaB p65 stimulates transcriptional
elongation.
J Virol
75
8524
8537
45. DudleyDMGaoYNelsonKNHenryKRNankyaI
2009
A novel yeast-based recombination method to clone and propagate
diverse HIV-1 isolates.
Biotechniques
46
458
467
46. FisherRABertonisJMMeierWJohnsonVACostopoulosDS
1988
HIV infection is blocked in vitro by recombinant soluble
CD4.
Nature
331
76
78
47. PugachPMarozsanAJKetasTJLandesELMooreJP
2007
HIV-1 clones resistant to a small molecule CCR5 inhibitor use the
inhibitor-bound form of CCR5 for entry.
Virology
361
212
228
48. de ClercqE
2010
In search of a selective therapy of viral
infections.
Antiviral Res
85
19
24
49. ArtsEJLiXGuZKleimanLParniakMA
1994
Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in
an endogenous human immunodeficiency virus-1 in vitro reverse
transcription/template-switching reaction.
J Biol Chem
269
14672
14680
50. RennerSLudwigVBodenOSchefferUGobelM
2005
New inhibitors of the Tat-TAR RNA interaction found with a
“fuzzy” pharmacophore model.
Chembiochem
6
1119
1125
51. HeMYuanDLinWPangRYuX
2005
Synthesis and assay of isoquinoline derivatives as HIV-1 Tat-TAR
interaction inhibitors.
Bioorg Med Chem Lett
15
3978
3981
52. MeiHYCuiMHeldsingerALemrowSMLooJA
1998
Inhibitors of protein-RNA complexation that target the RNA:
specific recognition of human immunodeficiency virus type 1 TAR RNA by small
organic molecules.
Biochemistry
37
14204
14212
53. BranchAD
1998
A good antisense molecule is hard to find.
Trends Biochem Sci
23
45
50
54. JacqueJMTriquesKStevensonM
2002
Modulation of HIV-1 replication by RNA
interference.
Nature
418
435
438
55. TamilarasuNHuqIRanaTM
2001
Targeting RNA with peptidomimetic oligomers in human
cells.
Bioorg Med Chem Lett
11
505
507
56. Sumner-SmithMZhengYZhangYPTwistEMClimieSC
1995
Antiherpetic activities of N-alpha-acetyl-nona-D-arginine amide
acetate.
Drugs Exp Clin Res
21
1
6
57. ApolloniAMeredithLWSuhrbierAKiernanRHarrichD
2007
The HIV-1 Tat protein stimulates reverse transcription in
vitro.
Curr HIV Res
5
473
483
58. HarrichDUlichCGarcia-MartinezLFGaynorRB
1997
Tat is required for efficient HIV-1 reverse
transcription.
EMBO J
16
1224
1235
59. HarrichDUlichCGaynorRB
1996
A critical role for the TAR element in promoting efficient human
immunodeficiency virus type 1 reverse transcription.
J Virol
70
4017
4027
60. BoudierCStorchakRSharmaKKDidierPFollenius-WundA
2010
The mechanism of HIV-1 Tat-directed nucleic acid annealing
supports its role in reverse transcription.
J Mol Biol
400
487
501
61. ZengYLiuHWLandesCFKimYJMaX
2007
Probing nucleation, reverse annealing, and chaperone function
along the reaction path of HIV-1 single-strand transfer.
Proc Natl Acad Sci U S A
104
12651
12656
62. BerkhoutBVastenhouwNLKlasensBIHuthoffH
2001
Structural features in the HIV-1 repeat region facilitate strand
transfer during reverse transcription.
RNA
7
1097
1114
63. DriscollMDHughesSH
2000
Human immunodeficiency virus type 1 nucleocapsid protein can
prevent self-priming of minus-strand strong stop DNA by promoting the
annealing of short oligonucleotides to hairpin sequences.
J Virol
74
8785
8792
64. GuoJHendersonLEBessJKaneBLevinJG
1997
Human immunodeficiency virus type 1 nucleocapsid protein promotes
efficient strand transfer and specific viral DNA synthesis by inhibiting
TAR-dependent self-priming from minus-strand strong-stop
DNA.
J Virol
71
5178
5188
65. IselCWesthofEMassireCLe GriceSFEhresmannB
1999
Structural basis for the specificity of the initiation of HIV-1
reverse transcription.
EMBO J
18
1038
1048
66. BaudinFMarquetRIselCDarlixJLEhresmannB
1993
Functional sites in the 5′ region of human immunodeficiency
virus type 1 RNA form defined structural domains.
J Mol Biol
229
382
397
67. PanCMezeiMMujtabaSMullerMZengL
2007
Structure-guided optimization of small molecules inhibiting human
immunodeficiency virus 1 Tat association with the human coactivator
p300/CREB binding protein-associated factor.
J Med Chem
50
2285
2288
68. Heilman-MillerSLWuTLevinJG
2004
Alteration of nucleic acid structure and stability modulates the
efficiency of minus-strand transfer mediated by the HIV-1 nucleocapsid
protein.
J Biol Chem
279
44154
44165
69. MeredithLWSivakumaranHMajorLSuhrbierAHarrichD
2009
Potent inhibition of HIV-1 replication by a Tat
mutant.
PLoS One
4
e7769
70. DasATHarwigAVrolijkMMBerkhoutB
2007
The TAR hairpin of human immunodeficiency virus type 1 can be
deleted when not required for Tat-mediated activation of
transcription.
J Virol
81
7742
7748
71. ChurcherMJLamontCHamyFDingwallCGreenSM
1993
High affinity binding of TAR RNA by the human immunodeficiency
virus type-1 tat protein requires base-pairs in the RNA stem and amino acid
residues flanking the basic region.
J Mol Biol
230
90
110
72. KeenNJChurcherMJKarnJ
1997
Transfer of Tat and release of TAR RNA during the activation of
the human immunodeficiency virus type-1 transcription elongation
complex.
EMBO J
16
5260
5272
73. AdachiAGendelmanHEKoenigSFolksTWilleyR
1986
Production of acquired immunodeficiency syndrome-associated
retrovirus in human and nonhuman cells transfected with an infectious
molecular clone.
J Virol
59
284
291
74. MarozsanAJTorreVSJohnsonMBallSCCrossJV
2001
Mechanisms involved in stimulation of human immunodeficiency
virus type 1 replication by aminooxypentane RANTES.
J Virol
75
8624
8638
75. HopeTJHuangXJMcDonaldDParslowTG
1990
Steroid-receptor fusion of the human immunodeficiency virus type
1 Rev transactivator: mapping cryptic functions of the arginine-rich
motif.
Proc Natl Acad Sci U S A
87
7787
7791
76. PageKALandauNRLittmanDR
1990
Construction and use of a human immunodeficiency virus vector for
analysis of virus infectivity.
J Virol
64
5270
5276
77. TorreVSMarozsanAJAlbrightJLCollinsKRHartleyO
2000
Variable sensitivity of CCR5-tropic human immunodeficiency virus
type 1 isolates to inhibition by RANTES analogs.
J Virol
74
4868
4876
78. ArtsEJStetorSRLiXRauschJWHowardKJ
1996
Initiation of (-) strand DNA synthesis from tRNA(3Lys) on
lentiviral RNAs: implications of specific HIV-1 RNA-tRNA(3Lys) interactions
inhibiting primer utilization by retroviral reverse
transcriptases.
Proc Natl Acad Sci U S A
93
10063
10068
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Hygiena a epidemiológia Infekčné lekárstvo LaboratóriumČlánok vyšiel v časopise
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