Online citations, reference lists, and bibliographies.
← Back to Search

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

Cite This
Download PDF
Analyze on Scholarcy
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
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)
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)
Review of HIV-1 reverse transcriptase three-dimensional structure : implications for drug design
R. G. Nanni (1993)
Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS).
F. Barré-Sinoussi (1983)
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)
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)
Nucleotide sequence of the AIDS virus, LAV
S. Wain-Hobson (1985)
Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion.
S. Lehrer (1971)
Localization of a polynucleotide binding region in the HIV-1 reverse transcriptase: implications for primer binding.
R. Sobol (1991)
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)
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)
Resonance energy transfer measurements between substrate binding sites within the large (Klenow) fragment of Escherichia coli DNA polymerase I.
D. Allen (1989)
Active human immunodeficiency virus protease is required for viral infectivity.
N. Kohl (1988)
Structure/function studies of HIV-1(1) reverse transcriptase: dimerization-defective mutant L289K.
R. Goel (1993)
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)
Kinetics of interaction of HIV reverse transcriptase with primer/template.
G. Divita (1993)
HIV-1 reverse transcriptase: crystallization and analysis of domain structure by limited proteolysis.
D. M. Lowe (1988)
Fluorescent oligonucleotides and deoxynucleotide triphosphates: preparation and their interaction with the large (Klenow) fragment of Escherichia coli DNA polymerase I.
D. Allen (1989)
Synthesis and application of derivatizable oligonucleotides.
K. Gibson (1987)
Kinetic and equilibrium intermediates in protein folding.
O. Ptitsyn (1994)
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)
Human immunodeficiency virus type 1 (HIV‐1) recombinant reverse transcriptase
S. K. Sharma (1994)
The structure of protein-protein recognition sites.
J. Janin (1990)
Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor.
L. A. Kohlstaedt (1992)
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)
Folding and association of proteins.
R. Jaenicke (1982)
Understanding how proteins fold: the lysozyme story so far.
C. Dobson (1994)
Fluorescence techniques for studying protein structure.
M. Eftink (1991)
Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed
A. Jacobo-Molina (1993)
Protein-protein interactions of HIV-1 reverse transcriptase: implication of central and C-terminal regions in subunit binding.
S. Becerra (1991)
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)
Structure of the binding site for nonnucleoside inhibitors of the reverse transcriptase of human immunodeficiency virus type 1.
S. Smerdon (1994)
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)
HIV inhibitors targeted at the reverse transcriptase.
E. Clercq (1992)
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)
Fluorescence quenching studies with proteins.
M. Eftink (1981)
Nonnucleoside inhibitors of HIV-1 reverse transcriptase: nevirapine as a prototype drug.
P. M. Grob (1992)
Interaction of fluorescently labeled dideoxynucleotides with HIV-1 reverse transcriptase.
B. Mueller (1991)

This paper is referenced by
Structural investigations on HIV-1 RT using single pair fluorescence resonance energy transfer
P. J. Rothwell (2002)
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)
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)
Inhibition of HIV‐1 Reverse Transcriptase Dimerization by Small Molecules
C. Tintori (2016)
NMR structure of the HIV-1 reverse transcriptase thumb subdomain
Naima G. Sharaf (2016)
MAPPIT (MAmmalian Protein-Protein Interaction Trap) as a tool to study HIV reverse transcriptase dimerization in intact human cells.
E. Pattyn (2008)
Selective unfolding of one Ribonuclease H domain of HIV reverse transcriptase is linked to homodimer formation
X. Zheng (2014)
Structural integrity of the ribonuclease H domain in HIV‐1 reverse transcriptase
Ryan L. Slack (2015)
Role of the "Helix Clamp" in HIV-1 Reverse Transcriptase Catalytic Cycling as Revealed by Alanine-scanning Mutagenesis (*)
W. Beard (1996)
Role of residues in the tryptophan repeat motif for HIV-1 reverse transcriptase dimerization.
G. Tachedjian (2003)
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)
Communicable Diseases of the Developing World
Anil Kumar Saxena (2018)
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)
Denaturation and reactivation of dimeric human glutathione reductase--an assay for folding inhibitors.
A. Nordhoff (1997)
TSAO compounds: the comprehensive story of a unique family of HIV-1 specific inhibitors of reverse transcriptase.
M. Camarasa (2004)
Targeting Small Molecules and Peptides to the p66-p51 Reverse Transcriptase Interface
Daouda Mousatpha Abba Moussa (2013)
Kinetics of association and dissociation of HIV-1 reverse transcriptase subunits.
C. F. Venezia (2009)
A New Potent HIV-1 Reverse Transcriptase Inhibitor
M. Morris (1999)
Destabilization of the HIV-1 reverse transcriptase dimer upon interaction with N-acyl hydrazone inhibitors.
N. Sluis-Cremer (2002)
Proteolytic processing and particle maturation.
V. Vogt (1996)
Small Molecule Inhibitors Targeting HIV‐1 Reverse Transcriptase Dimerization
Dina Grohmann (2008)
Human Immunodeficiency Virus Reverse Transcriptase
M. Wendeler (2009)
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)
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)
Targeting HIV-1 integrase with aptamers selected against the purified RNase H domain of HIV-1 RT.
M. Métifiot (2005)
The p66 immature precursor of HIV‐1 reverse transcriptase
Naima G. Sharaf (2014)
Modulation of the oligomeric structures of HIV-1 retroviral enzymes by synthetic peptides and small molecules.
N. Sluis-Cremer (2002)
See more
Semantic Scholar Logo Some data provided by SemanticScholar