p66 Trp24 and Phe61 Are Essential for Accurate Association of HIV-1 Reverse Transcriptase with Primer/Template

doi:10.1016/j.jmb.2007.07.044

Journal of Molecular Biology

Volume 373, Issue 1, 12 October 2007, Pages 127-140

https://doi.org/10.1016/j.jmb.2007.07.044 Get rights and content

Abstract

Preventing dimerization of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) constitutes an alternative strategy to abolish virus proliferation. We have previously demonstrated that a short peptide derived from the Trp cluster of the connection domain disrupts the RT dimer by interacting with Trp24 and Phe61 in a cleft located between the fingers and the connection domains of p51. Both Trp24 and Phe61 of p51 are essential for the stability of the RT dimer. Here, in order to understand the requirement of Trp24 and Phe61 in the p66 subunit, we have investigated their implication in the formation of RT–primer/template (p/t) complexes and in RT processivity by combining pre-steady-state and steady-state kinetics with site-directed mutagenesis. We demonstrate that both residues are essential for proper binding of the p/t and control conformational changes required for RT ordered mechanism. Trp24 and Phe61 act on p/t binding and remodeling of the catalytic site. Phe61G mutation increases the binding “on” rate of both p/t and mismatched p/t, yielding an unfavorable RT–p/t for polymerase catalysis, unable to pursue mispair extension. Considering the structure of unliganded RT, Phe61 seems to be involved in the dynamics of p66 thumb–finger interactions and in stabilization of the p/t in the catalytic site. In contrast, the p66 Trp24G mutation alters the overall kinetics of p/t binding and is essentially involved in stabilizing the RT–p/t complex by contacting the 5′ overhang of the template strand. Mutation of both Trp24 and Phe61 alters mispair extension efficiency, suggesting that disruption of the tight contacts between the fingers domain and the 5′ overhang of the template strand increases RT fidelity and reduces RT processivity. Taken together, these studies infer that mutations altering the aromatic nature of Phe61 or Trp24 that may occur to counteract peptide inhibitors targeting this region will generate an unstable RT exhibiting low polymerase activity and higher fidelity. As such, our work suggests that the combined application of peptide-based RT dimerization inhibitors is likely to be highly efficient.

Introduction

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) plays an essential role in HIV replication. RT is a multifunctional enzyme responsible for both RNA- and DNA-dependent DNA polymerase and RNase-H activities required for the conversion of single-stranded viral RNA into double-stranded DNA.¹^,² RT constitutes one of the main targets for therapeutic treatment of AIDS; however, the major limitation of RT inhibitors currently administered in the clinic, including both nucleoside RT and nonnucleoside RT inhibitors (NNRTI), is the rapid emergence of resistant strains.3., 4., 5. Together, the poor fidelity of DNA polymerase and the high level of errors made by HIV-1 RT, associated with the important viral replication rate, are responsible for the high genetic variation of HIV and therefore the rapid emergence of drug resistance.⁶^,⁷

In order to offer new perspectives for the design of RT inhibitors, extensive efforts have been made in the design of molecules that target the structural organization of the enzyme.8., 9., 10. The biologically active form of RT is an asymmetric heterodimer containing two subunits, p66 and p51, each composed of four common subdomains called palm, fingers, thumb and connection and an RNase-H domain; the latter presents only on the larger subunit.¹¹^,¹² The small p51 subunit is derived from p66 by proteolytic cleavage of the C-terminal RNase-H domain. As such, both p66 and p51 harbor a polymerase domain, but these individual subunits are catalytically inert as monomers, dimeric structure being a prerequisite for activation of both polymerase and RNase-H activities.13., 14., 15., 16., 17. We¹⁰^,¹⁸ and others¹⁹^,²⁰ have postulated that the heterodimeric organization of RT constitutes an interesting target for the design of new inhibitors and have demonstrated that preventing or controlling RT dimerization is an alternative to blocking HIV proliferation and has a major impact on the viral cycle.¹⁹^,²⁰ We have developed a new generation of inhibitors based on a short synthetic decapeptide, “Pep-7,” derived from the Trp cluster of the connection subdomain, which mimics the protein interface and disrupts and prevents protein–protein interactions.10., 18., 20. This inhibitor binds a cleft located between the connection and the fingers domains of the p51 subunit and forms a major interaction with the highly conserved residues Trp24 and Phe61 of p51, which play a central role in Pep-7 inhibitory mechanism.²¹ We have demonstrated that a mutation altering the aromatic character of these two residues on p51 partially abolishes binding of Pep-7 to RT, while also significantly reducing the stability of the heterodimer.²¹ As p51 derives from p66, mutations of Trp24 and of Phe61 occurring in vivo will automatically be found on both subunits. It is therefore of major interest to understand the impact of these mutations on p66 on RT integrity and its enzymatic mechanism.

HIV-1 RT polymerase activity has been largely characterized and follows an ordered mechanism involving, first, binding of the primer/template (p/t), followed by deoxynucleoside triphosphate (dNTP) binding, which triggers a conformational change leading to attack of the 3′ OH of the primer terminus on the dNTP alpha phosphorus and formation of a product complex.22., 23., 24., 25. The conformational change constitutes the rate-limiting step of the mechanism, which is followed by elongation or release of the elongated p/t. Mechanistic investigations together with the determination of RT structures complexed¹¹^,¹²^,²⁶ or not²⁷^,²⁸ with substrates or inhibitors⁷^,¹²^,²⁵^,²⁹^,³⁰ have provided information on the conformational changes that occur during polymerization and which are associated with inhibitor and drug resistance. Residue Phe61 is located in the β3–β4 hairpin of the fingers domain of p66 and forms key contacts for the templating nucleotide and the incoming dNTP.²⁶ This residue has been reported to be involved in strand displacement DNA synthesis and in control of the incoming template strand and RT fidelity.³²^,³³ In general, residues in the β3–β4 loop hairpin of the fingers subdomain of p66 have been reported to control several aspects of RT activity, including fidelity, processivity, pyrophosphorolysis and strand displacement synthesis and harbor hotspot residues for nucleoside analog resistance mutations.²⁶^,32., 33., 34., 35. In contrast, the role of Trp24 in p66 on RT polymerase activity and on the stability of the RT–p/t complex is poorly documented. This residue contacts nucleosides + 3 and + 2 of the template strand as shown in the structure of RT-trapped p/t.²⁶ In the present work, we have combined steady-state and pre-steady-state approaches to better characterize the role of p66 Trp24 and Phe61 in formation of the RT–p/t complex and in both RT processivity and fidelity. We demonstrate that mutation of both residues into Gly decreases the stability of the RT–p/t complex without affecting binding of the incoming dNTP. Both Trp24 and Phe61 promote proper binding of p/t to RT, but act on the accuracy of binding in different fashions. Taken together, these structural and kinetic investigations provide a better insight into the role of Trp24 and Phe61 residues of the p66 subunits of HIV-1 RT.

Section snippets

Mutations of Trp24 and Phe61 on p66 affect the polymerase activity of RT

We have previously demonstrated that both Trp24 and Phe61 of the p51 subunit play an important role in the stabilization of the RT heterodimer, but are not involved in its catalytic activities.²¹ Here, we have investigated the impact of both Trp24 and Phe61 of the p66 subunit on RT polymerase activity. To assess the influence of these residues, we first analyzed to what extent single Trp24Gly, Phe61Gly and double Trp24Gly-Phe61Gly mutations on the p66 subunit could alter RT polymerase activity

Discussion

The heterodimeric architecture of HIV-1 RT can be altered by small peptides derived from the Trp cluster of the connection subdomains that mimic interface domains and therefore constitute potent antiviral molecules.¹⁰^,²⁰ The binding of inhibitory peptides occurs in a cleft between the connection and the fingers subdomains of the p51 subunit, and mainly involves interactions with the highly conserved Trp24 and Phe61 residues.²¹ In order to evaluate the potency of mutations of these two residues

Materials

[α-³²P]dATP, [α-³²P]dTTP, dGTP and dTTP were purchased from GE Healthcare Europe GmbH (Orsay, France). Primer and template oligodeoxynucleotides were purchased from MWG Biotech AG (Ebersberg, Germany). The sequences of the 19/36-mer DNA/DNA p/t used for fluorescence titration and stopped-flow experiments were 5′-TCCCTGTTCGGGCGCCACT-3′ and 5′-TGTGGAAAATCTCATGCAGTGGCGCCCGAACAGGGA-3′, respectively (Table 2). The primer was labeled at the 3′ end with FAM on the thymine base. The complementary

Acknowledgements

This work was supported in part by the Centre National de la Recherche Scientifique (CNRS) and by grants from the Agence Nationale de Recherche sur le SIDA (ANRS), SIDACTION and the Fondation pour la Recherche Médicale (FRM). A.A. and J.D. were supported by fellowships of the European Community and ANRS, respectively. This work is part of the program “Targeting Replication and Integration of HIV” (TRIoH) supported by the EC (LSHB-CT-2003-503480). We would like to thank R. Goody and T. Restle

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During reverse transcription of the HIV-1 genome, two strand-transfer events occur. Both events rely on the RNase H cleavage activity of reverse transcriptases (RTs) and template homology. Using a panel of mutants of HIV-1_BH10 (group M/subtype B) and HIV-1_ESP49 (group O) RTs and in vitro assays, we demonstrate that there is a strong correlation between RT minus-strand transfer efficiency and template-primer binding affinity. The highest strand transfer efficiencies were obtained with HIV-1_ESP49 RT mutants containing the substitutions K358R/A359G/S360A, alone or in combination with V148I or T355A/Q357M. These HIV-1_ESP49 RT mutants had been previously engineered to increase their DNA polymerase activity at high temperatures. Now, we found that RTs containing RNase H-inactivating mutations (D443N or E478Q) were devoid of strand transfer activity, whereas enzymes containing F61A or L92P had very low strand transfer activity. The strand transfer defect produced by L92P was attributed to a loss of template-primer binding affinity and, more specifically, to the higher dissociation rate constants (k_off) shown by RTs bearing this substitution. Although L92P also deleteriously affected the RT's nontemplated nucleotide addition activity, neither nontemplated nucleotide addition activity nor the RT's clamp activities contributed to increased template switching when all tested mutant and WT RTs were considered. Interestingly, our results also revealed an association between efficient strand transfer and the generation of secondary cleavages in the donor RNA, consistent with the creation of invasion sites. Exposure of the elongated DNA at these sites facilitate acceptor (RNA or DNA) binding and promote template switching.
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The reverse transcriptase (RT) of HIV (human immunodeficiency virus) is a key enzyme in the virus replication cycle and catalyzes a chain of reactions to convert the single-stranded HIV RNA genome into a double-stranded DNA for further integration in the cell host genome. This requires that RT is able to discriminate between different nucleic acids and to place them correctly in one of the three catalytic sites for RNA-dependent DNA synthesis, DNA-dependent DNA synthesis, and RNAse-H. RT structure and mechanism have been investigated in detail by combining steady-state transient kinetic, and FRET single-molecule assay using dyes attached to the enzyme or to its different partners: tRNA, viral-RNA, and DNA primer/template.38–41 These investigations have shown that catalysis of RT is dependent on the binding orientation of the substrate, which adopts opposite conformations for DNA and RNA duplexes (Fig. 4.9).
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Mechanistic insights into the roles of three linked single-stranded template binding residues of MMLV reverse transcriptase in misincorporation and mispair extension fidelity of DNA synthesis
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In summary, these template binding sites (Y64, D114, and R116 in MMLV RT; W24, D76, and R78 in HIV-1 RT) binding to the extended single-stranded template backbone share similar topology and some similar interactions in MMLV RT and HIV-1 RT, such as aromatic stacking between aromatic residue Y64 (W24 in HIV-1 RT) and template nucleotides, interaction of a negatively charged D114 (D76 in HIV-1 RT) to template n and ion-pairing between D114 and R116 (D76–R78 in HIV-1 RT). These similarities confer them with some similar displays in their polymerase activity, gel-shift, and processivity assays (Agopian et al., 2007; Depollier et al., 2005; Kim et al., 1998, 1999). On the other hand, the above-mentioned characteristics in this structural set of MMLV RT presumably assist in the formation of an accurate nascent base pair binding pocket, thereby supplying MMLV RT with the highest fidelity among RTs studied.
To obtain some insights into the structure–function relationship of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT), we modeled the catalytic state ternary complexes of this protein using the corresponding RT from human immunodeficiency virus type 1 (HIV-1) and available structures of MMLV RT. We observed that three MMLV RT single-stranded template binding residues, Y64, D114, and R116, act as a linked set through mutual interactions, including hydrogen bonding and ion-pairing. The analogous residues of HIV-1 RT have a somewhat different environment and they lack this linked phenomenon. To understand the functional implication of this linked set of MMLV RT, we performed site-directed mutagenesis at these three positions. Then the mutant enzymes were examined for their biochemical properties and nucleotide selectivity. Mutagenesis of these residues (Y64A, D114A, and R116A) resulted in enzymes with slight to modest changes in polymerase activities. The processivity of DNA synthesis correlated positively with the binding affinity of the MMLV RT variants. Lower fidelity in mutants was indicated by measurements of misincorporation and mispair extension fidelity of wild type (WT) and mutant RTs, in contrast to earlier works that indicate that mutations at the analogous positions in HIV-1 RT result in relatively higher fidelity. These data together with structural analysis suggest that this structural set may therefore be a key factor responsible for the different fidelity of these two RTs.
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Lys65 residue, in the fingers domain of human immunodeficiency virus reverse transcriptase (RT), interacts with incoming dNTP in a sequence-independent fashion. We showed previously that a 5-amino-acid deletion spanning Lys65 and a K65A substitution both enhanced the fidelity of dNTP insertion. We hypothesized that the Lys65 residue enhances dNTP misinsertion via interactions with the γ-phosphate of the incoming dNTP. We now examine this hypothesis in pre-steady-state kinetic studies using wild-type human immunodeficiency virus-1 RT and two substitution mutants, K65A and K65R. K65R mutation did not greatly increase misinsertion fidelity, but K65A mutation led to higher incorporation fidelity. For a misinsertion to become a permanent error, it needs to be accompanied by the extension of the mispaired terminus thus formed. Both mutants and the wild-type enzyme discriminated against the mismatched primer at the catalytic step (k_pol). Additionally, K65A and K65R mutants displayed a further decrease in mismatch extension efficiency, primarily at the level of dNTP binding. We employed hydroxyl radical footprinting to determine the position of the RT on the primer/template. The wild-type and Lys65-substituted enzymes occupied the same position at the primer terminus; the presence of a mismatched primer terminus caused all three enzymes to be displaced to a − 2 position relative to the primer 3′ end. In the context of an efficiently extended mismatched terminus, the presence of the next complementary nucleotide overcame the displacement, resulting in a complex resembling the matched terminus. The results are consistent with the observed reduction in k_pol in mispaired primer extension being due to the position of the enzyme at a mismatched terminus. Our work shows the influence of the stabilizing interactions of Lys65 with the incoming dNTP on two different aspects of polymerase fidelity.
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The 5.6-fold lower Kd of labeled FITC-PAW over unlabeled peptide suggests that the dye contacts RT and stabilizes the peptide within its binding site. Because both Trp24 and Phe61 located on the fingers domain of p66 subunit have been reported to be involved in the control of p/t binding and in the dynamics of the thumb-fingers subdomain interactions (34, 44, 45), we then evaluated the binding of PAW on RT harboring single Phe61Gly and double Phe61Gly and Trp24Gly mutations on the p66 subunit. In comparison to wild-type RT, the affinity of PAW was reduced 6-fold (Kd: 207 ± 62 nm) for p66F61G/p51wt and 4.5-fold (Kd: 149 ± 38 nm) for p66DM/p51wt (Fig. 3A).
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^†: A.A. and J.D. contributed equally to this work.

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Journal of Molecular Biology

p66 Trp24 and Phe61 Are Essential for Accurate Association of HIV-1 Reverse Transcriptase with Primer/Template

Abstract

Introduction

Section snippets

Mutations of Trp24 and Phe61 on p66 affect the polymerase activity of RT

Discussion

Materials

Acknowledgements

Trends Pharmacol. Sci.

Curr. Opin. Struct. Biol.

Antiviral Res.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

FEBS Letters

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Structure

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

Reverse transcriptase and the generation of retroviral DNA

Human immunodeficiency virus reverse transcriptase

Antiretroviral drug resistance: mechanisms, pathogenesis, clinical significance

Adv. Exp. Med. Biol.

Inhibitors of HIV-1 reverse transcriptase

Adv. Pharmacol.

Strategies in the design of antiviral drugs

Nat. Rev. Drug Discov.