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Dimerization Kinetics Of HIV-1 And HIV-2 Reverse Transcriptase: A Two Step Process.

G. Divita, K. Rittinger, C. Geourjon, G. Deléage, R. Goody
Published 1995 · Chemistry, Medicine

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The dimerization processes of the human immunodeficiency virus (HIV) types 1 and 2 reverse transcriptase (RTs) from their subunits have been investigated using a number of complementary approaches (fluorescence spectroscopy, size exclusion-HPLC and polymerase activity assay). The formation of the native heterodimeric form of HIV-1 and HIV-2 RT occurs in a two step process. The first step is a concentration-dependent association of the two subunits (p66 and p51) to give a heterodimeric intermediate, which slowly isomerizes to the "mature" heterodimeric form of the enzyme. For both RTs, the first step behaves as a second order reaction with similar association rate constants (in the range of 2 x 10(4) to 4 x 10(4) M-1 s-1). This initial dimerization results in a 25% quenching of the intrinsic fluorescence and a 30% decrease in the accessibility of the tryptophan hydrophobic cluster to solvent as revealed by iodide quenching experiments and by monitoring the binding of 1-anilino-8-naphthalenesulphonate. The formation of the intermediate-RT form appears to involve hydrophobic regions of the subunits containing tryptophan residues. This intermediate form is devoid of polymerase activity, but is able to bind primer/template with high affinity. The final stage of the mature RT-heterodimer formation occurs in a slow first order reaction, which is 12-fold faster for HIV-2 (1.2 h-1) than HIV-1 RT (0.1 h-1). At micromolar concentrations, this slow isomerization constitutes the rate limiting step of the RT maturation and the structural change involved appears to be partly associated with the catalytic site, as shown using fluorescent labelled primer/template. On the basis of both the presently available X-ray structure of the HIV-1 RT and the predicted structure of HIV-2 RT, the thumb subdomain of the p51 subunit seems to be involved in this maturation step, which is probably the interaction of this domain with the RNAse H domain of the large subunit. The placement of the fingers subdomain of p51 in the palm subdomain of the p66 subunit may also be associated with formation of mature heterodimeric RTs.
This paper references
10.1016/0014-5793(92)80172-D
RNase H activity of HIV reverse transcriptases is confined exclusively to the dimeric forms
T. Restle (1992)
JMB — MS 296 Dimerization of HIV Reverse Transcriptase 520 utilizing the principle of proteindye binding
E. De Clercq (1992)
10.1021/BI00240A001
HIV reverse transcriptase structure-function relationships.
A. Jacobo-Molina (1991)
Isolation of a Tlymphotropic retrovirus from a patient at risk for acquired immunodeficiency syndrome ( AIDS )
F. Barré-Sinoussi (1983)
Refolding and association of oligomeric proteins.
R. Jaenicke (1986)
10.1007/BF02171659
Review of HIV-1 reverse transcriptase three-dimensional structure : implications for drug design
R. G. Nanni (1993)
10.1126/SCIENCE.6189183
Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS).
F. Barré-Sinoussi (1983)
10.1126/SCIENCE.2418504
Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV.
F. di Marzo Veronese (1986)
Structure-function relationships of HIV-1 reverse transcriptase determined using monoclonal antibodies.
T. Restle (1992)
Measuring the conformation stability of a protein
C. N. Pace (1990)
10.1126/SCIENCE.6200935
Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS.
M. Popovič (1984)
A rapid and sensitive method for the quantitation of microgram quantities of protein
M. Bradford (1976)
10.1016/0092-8674(85)90303-4
Nucleotide sequence of the AIDS virus, LAV
S. Wain-Hobson (1985)
10.1021/BI00793A015
Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion.
S. Lehrer (1971)
10.1021/BI00108A004
Localization of a polynucleotide binding region in the HIV-1 reverse transcriptase: implications for primer binding.
R. Sobol (1991)
10.1016/0014-5793(93)81383-B
Characterization of the dimerization process of HIV‐1 reverse transcriptase heterodimer using intrinsic protein fluorescence
G. Divita (1993)
Expression of the heterodimeric form of human immunodeficiency virus type 2 reverse transcriptase in Escherichia coli and characterization of the enzyme.
B. Mueller (1991)
10.1073/PNAS.90.13.6320
Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA.
A. Jacobo-Molina (1993)
10.1021/BI00451A006
Resonance energy transfer measurements between substrate binding sites within the large (Klenow) fragment of Escherichia coli DNA polymerase I.
D. Allen (1989)
10.1073/PNAS.85.13.4686
Active human immunodeficiency virus protease is required for viral infectivity.
N. Kohl (1988)
10.1021/BI00211A009
Structure/function studies of HIV-1(1) reverse transcriptase: dimerization-defective mutant L289K.
R. Goel (1993)
10.1016/S0006-3495(94)80799-4
The use of fluorescence methods to monitor unfolding transitions in proteins.
M. Eftink (1994)
JMB — MS 296 Dimerization of HIV Reverse Transcriptase 520 utilizing the principle of proteindye binding
E. De Clercq (1992)
10.1021/BI00082A018
Kinetics of interaction of HIV reverse transcriptase with primer/template.
G. Divita (1993)
10.1021/BI00425A002
HIV-1 reverse transcriptase: crystallization and analysis of domain structure by limited proteolysis.
D. M. Lowe (1988)
10.1021/BI00437A014
Fluorescent oligonucleotides and deoxynucleotide triphosphates: preparation and their interaction with the large (Klenow) fragment of Escherichia coli DNA polymerase I.
D. Allen (1989)
10.1093/NAR/15.16.6455
Synthesis and application of derivatizable oligonucleotides.
K. Gibson (1987)
10.1093/PROTEIN/7.5.593
Kinetic and equilibrium intermediates in protein folding.
O. Ptitsyn (1994)
10.1126/SCIENCE.2983426
Rapid and sensitive protein similarity searches.
D. Lipman (1985)
Co-expression of the subunits of the heterodimer of HIV-1 reverse transcriptase in Escherichia coli.
B. Müller (1989)
Dimerization der gag-pol- und pol-Polyproteine von HIV-1 und Aspekte der polProzessierung in zellfreien Translationssystemen. M.D
M. Gautel (1990)
10.1016/0014-5793(94)80303-X
Human immunodeficiency virus type 1 (HIV‐1) recombinant reverse transcriptase
S. K. Sharma (1994)
The structure of protein-protein recognition sites.
J. Janin (1990)
10.1126/SCIENCE.1377403
Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor.
L. A. Kohlstaedt (1992)
10.1093/PROTEIN/7.2.157
SOPM: a self-optimized method for protein secondary structure prediction.
C. Geourjon (1994)
HIV reverse transcriptase
A. Jacobo-Molina (1991)
Resonance energy transfer measurements between substrate binding sites within the large (Klenow) fragment of Escherichia coli
D. J. Allen (1989)
10.1016/0079-6107(87)90011-3
Folding and association of proteins.
R. Jaenicke (1982)
10.1016/0968-0004(94)90171-6
Understanding how proteins fold: the lysozyme story so far.
C. Dobson (1994)
10.1002/9780470110560.CH3
Fluorescence techniques for studying protein structure.
M. Eftink (1991)
Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed
A. Jacobo-Molina (1993)
10.1021/BI00114A015
Protein-protein interactions of HIV-1 reverse transcriptase: implication of central and C-terminal regions in subunit binding.
S. Becerra (1991)
10.1021/BI00117A045
Identification of the primer binding domain in human immunodeficiency virus reverse transcriptase.
A. Basu (1992)
Inhibition of human immunodeficiency virus type 1 reverse transcriptase dimerization using synthetic peptides derived from the connection domain.
G. Divita (1994)
10.1073/PNAS.91.9.3911
Structure of the binding site for nonnucleoside inhibitors of the reverse transcriptase of human immunodeficiency virus type 1.
S. Smerdon (1994)
10.1128/JVI.60.2.771-775.1986
Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus.
M. Lightfoote (1986)
Modulation of HIV-1 reverse transcriptase function in "selectively deleted" p66/p51 heterodimers.
P. Jacques (1994)
10.1089/AID.1992.8.119
HIV inhibitors targeted at the reverse transcriptase.
E. Clercq (1992)
10.1016/0014-5793(90)80143-7
Evidence for a molten globule state as a general intermediate in protein folding
O. Ptitsyn (1990)
A leucine zipper-like motif may mediate HIV reverse transcriptase subunit binding.
J. Baillon (1991)
10.1016/0003-2697(81)90474-7
Fluorescence quenching studies with proteins.
M. Eftink (1981)
10.1089/AID.1992.8.145
Nonnucleoside inhibitors of HIV-1 reverse transcriptase: nevirapine as a prototype drug.
P. M. Grob (1992)
10.1021/BI00229A017
Interaction of fluorescently labeled dideoxynucleotides with HIV-1 reverse transcriptase.
B. Mueller (1991)



This paper is referenced by
10.17877/DE290R-15321
Structural investigations on HIV-1 RT using single pair fluorescence resonance energy transfer
P. J. Rothwell (2002)
10.1517/13543784.5.8.985
Therapeutic potential of nonnucleoside reverse transcriptase inhibitors in the treatment of HIV infection
R. A. Spence (1996)
Characterization of the mechanism of action of new HIV-1 reverse transcriptase-associated ribonuclease H inhibitors
A. Corona (2014)
10.1177/095632020501600301
TSAO Derivatives the First Non-Peptide Inhibitors of HIV-1 RT Dimerization
M. Camarasa (2005)
Solution NMR studies of HIV-1 reverse transcriptase
Naima G. Sharaf (2017)
10.1002/cbic.201500668
Inhibition of HIV‐1 Reverse Transcriptase Dimerization by Small Molecules
C. Tintori (2016)
10.1007/s10858-016-0077-2
NMR structure of the HIV-1 reverse transcriptase thumb subdomain
Naima G. Sharaf (2016)
10.1016/j.jviromet.2008.06.021
MAPPIT (MAmmalian Protein-Protein Interaction Trap) as a tool to study HIV reverse transcriptase dimerization in intact human cells.
E. Pattyn (2008)
10.1093/nar/gku143
Selective unfolding of one Ribonuclease H domain of HIV reverse transcriptase is linked to homodimer formation
X. Zheng (2014)
10.1002/prot.24843
Structural integrity of the ribonuclease H domain in HIV‐1 reverse transcriptase
Ryan L. Slack (2015)
10.1074/jbc.271.21.12213
Role of the "Helix Clamp" in HIV-1 Reverse Transcriptase Catalytic Cycling as Revealed by Alanine-scanning Mutagenesis (*)
W. Beard (1996)
10.1016/S0022-2836(02)01433-X
Role of residues in the tryptophan repeat motif for HIV-1 reverse transcriptase dimerization.
G. Tachedjian (2003)
10.1016/J.JMB.2005.03.057
Identification of amino acid residues in the human immunodeficiency virus type-1 reverse transcriptase tryptophan-repeat motif that are required for subunit interaction using infectious virions.
Alok Mulky (2005)
10.1007/978-3-319-78254-6
Communicable Diseases of the Developing World
Anil Kumar Saxena (2018)
10.1093/NAR/25.14.2730
A new peptide vector for efficient delivery of oligonucleotides into mammalian cells.
M. Morris (1997)
Proteolytic cleavage events in the maturation of HIV-1 reverse transcriptase
M. E. Abram (2005)
Interacciones proteína-proteína como diana terapéutica en la transcriptasa interna del VIH-1 y en la tripanotión reductasa de "leishmania infantum"
S. Murcia (2013)
10.1111/J.1432-1033.1997.00273.X
Denaturation and reactivation of dimeric human glutathione reductase--an assay for folding inhibitors.
A. Nordhoff (1997)
10.2174/1568026043388600
TSAO compounds: the comprehensive story of a unique family of HIV-1 specific inhibitors of reverse transcriptase.
M. Camarasa (2004)
10.1007/978-1-4614-7291-9_8
Targeting Small Molecules and Peptides to the p66-p51 Reverse Transcriptase Interface
Daouda Mousatpha Abba Moussa (2013)
10.1021/bi9010495
Kinetics of association and dissociation of HIV-1 reverse transcriptase subunits.
C. F. Venezia (2009)
10.1074/jbc.274.35.24941
A New Potent HIV-1 Reverse Transcriptase Inhibitor
M. Morris (1999)
10.1124/MOL.62.2.398
Destabilization of the HIV-1 reverse transcriptase dimer upon interaction with N-acyl hydrazone inhibitors.
N. Sluis-Cremer (2002)
10.1007/978-3-642-80145-7_4
Proteolytic processing and particle maturation.
V. Vogt (1996)
10.1002/cbic.200700669
Small Molecule Inhibitors Targeting HIV‐1 Reverse Transcriptase Dimerization
Dina Grohmann (2008)
10.1007/B135974_19
Human Immunodeficiency Virus Reverse Transcriptase
M. Wendeler (2009)
10.1124/MOL.61.2.400
Chimeric human immunodeficiency virus type 1 and feline immunodeficiency virus reverse transcriptases: role of the subunits in resistance/sensitivity to non-nucleoside reverse transcriptase inhibitors.
J. Auwerx (2002)
This thesis is based on the following papers, which are referred to in the text by their Roman numerals.
I. Jonasson (2009)
10.1021/JM001095I
Identification of a putative binding site for [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)thymine (TSAO) derivatives at the p51-p66 interface of HIV-1 reverse transcriptase.
F. Rodríguez-Barrios (2001)
10.1016/J.BIOCHI.2005.03.013
Targeting HIV-1 integrase with aptamers selected against the purified RNase H domain of HIV-1 RT.
M. Métifiot (2005)
10.1002/prot.24594
The p66 immature precursor of HIV‐1 reverse transcriptase
Naima G. Sharaf (2014)
10.1046/J.1432-1033.2002.03216.X
Modulation of the oligomeric structures of HIV-1 retroviral enzymes by synthetic peptides and small molecules.
N. Sluis-Cremer (2002)
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