Dimeric HIV-1 Research Articles

Stability and conformation of the dimeric HIV-1 genomic RNA 5′UTR

During HIV-1 assembly, the viral Gag polyprotein specifically selects the dimeric RNA genome for packaging into new virions. The 5′ untranslated region (5′UTR) of the dimeric genome may adopt a conformation that is optimal for recognition by Gag. Further conformational rearrangement of the 5′UTR, promoted by the nucleocapsid (NC) domain of Gag, is predicted during virus maturation. Two 5′UTR dimer conformations, the kissing dimer (KD) and the extended dimer (ED), have been identified in vitro, which differ in the extent of intermolecular basepairing. Whether 5′UTRs from different HIV-1 strains with distinct sequences have access to the same dimer conformations has not been determined. Here, we applied fluorescence cross-correlation spectroscopy and single-molecule Förster resonance energy transfer imaging to demonstrate that 5′UTRs from two different HIV-1 subtypes form (KDs) with divergent stabilities. We further show that both 5′UTRs convert to a stable dimer in the presence of the viral NC protein, adopting a conformation consistent with extensive intermolecular contacts. These results support a unified model in which the genomes of diverse HIV-1 strains adopt an ED conformation.

6,7-Dihydroxyisoindolin-1-one and 7,8-Dihydroxy-3,4-Dihydroisoquinolin- 1(2H)-one Based HIV-1 Integrase Inhibitors.

Integrase (IN) is an essential viral enzyme required for HIV-1 replication, which has been targeted by anti-AIDS therapeutics. Integrase strand transfer inhibitors (INSTIs) represent a new class of antiretroviral agents developed for the treatment of HIV-1 infections. Important structural features that are shared by many INSTIs include a coplanar arrangement of three heteroatoms that chelate two catalytic Mg(2+) ions in the IN active site and a linked halophenyl ring that binds in the hydrophobic pocket formed by the complex of IN with viral DNA. We recently reported bicyclic 6,7-dihydroxyoxoisoindolin-1-one-based IN inhibitors. In the current study, we modified these inhibitors in three ways. First, we increased the spacer length between the metalchelating triad and the halophenyl group. Second, we replaced the indoline [5,6] bicycle with a fused dihydroxyisoquinolinones [6,6] ring system. Finally, we prepared bis-6,7-dihydroxyisoindolin-1-one-4-sulfonamides as dimeric HIV-1 IN inhibitors. These new analogues showed low micromolar inhibitory potency in in vitro HIV-1 integrase assays.

How Does Darunavir Prevent HIV-1 Protease Dimerization?

The drug Darunavir (DRV) is a potent inhibitor of HIV-1 protease (PR), a homodimeric essential enzyme of the AIDS virus. Recent experimental data suggest that DRV is able to prevent dimerization of HIV-1 PR, which, together with its high affinity for the mature enzyme, has been linked to the high genetic barrier to the development of viral resistance. The mechanism of dimerization inhibition and the binding mode(s) of DRV to monomeric HIV-1 PR are unknown. Here, multiple molecular dynamics simulations with explicit solvent (for a total of 11 μs with the CHARMM force field and 1 μs with the Amber force field) show that the monomer of HIV-1 PR is structurally stable and reveal a major binding mode of DRV. This binding mode is stabilized by favorable interactions between the apolar groups of DRV and the hydrophobic residues Ile32, Ile47, Ile50, Ile54, Pro79, Val82, and Ile84. The binding mode to monomeric HIV-1 PR identified by molecular dynamics is different from the two binding modes observed in the crystal structure of the complex with dimeric HIV-1 PR. As an example, there are no interactions between DRV and the catalytic Asp25 in the binding mode to monomeric HIV-1 PR revelead by the simulations. In contrast, the simulations show extensive and stable interactions between DRV and the flap (residues 46-55), which are likely to sterically hinder the formation of the flap interface as observed in the dimeric structure. Which of the two mechanisms of inhibition (dimerization inhibition by association with the flap or binding to the active site of the mature enzyme) dominates might depend on the HIV-1 PR mutations, and it is likely that dimerization inhibition is predominant for multiple mutations at the active site in multidrug resistant strains.

Using the Experimentally Determined Components of the Overall Rotational Diffusion Tensor To Restrain Molecular Shape and Size in NMR Structure Determination of Globular Proteins and Protein−Protein Complexes

This paper describes an approach for making use of the components of the experimentally determined rotational diffusion tensor derived from NMR relaxation measurements in macromolecular structure determination. The parameters of the rotational diffusion tensor describe the shape and size of the macromolecule or macromolecular complex, and are therefore complementary to traditional NMR restraints. The structural information contained in the rotational diffusion tensor is not dissimilar to that present in the small-angle region of solution X-ray scattering profiles. We demonstrate the utility of rotational diffusion tensor restraints for protein structure refinement using the N-terminal domain of enzyme I (EIN) as an example and validate the results by solution small-angle X-ray scattering. We also show how rotational diffusion tensor restraints can be used for docking complexes using the dimeric HIV-1 protease and the EIN-HPr complexes as examples. In the former case, the rotational diffusion tensor restraints are sufficient in their own right to determine the position of one subunit relative to another. In the latter case, rotational diffusion tensor restraints complemented by highly ambiguous distance restraints derived from chemical shift perturbation mapping and a hydrophobic contact potential are sufficient to correctly dock EIN to HPr. In each case, the cluster containing the lowest-energy structure corresponds to the correct solution.

An alternative strategy for inhibiting multidrug-resistant mutants of the dimeric HIV-1 protease by targeting the subunit interface

Mutations that occur in response to the HIV-1 protease inhibitors are responsible for the development of multidrug cross-resistance to these antiproteases in AIDS treatment. One alternative to inhibiting the active site of HIV-1 protease is to target the dimer interface of the homodimeric enzyme at the antiparallel beta-sheet formed by the interdigitation of the C- and N-ends of each monomer. This region is highly conserved and is responsible for approx. 75% of the dimer-stabilization energy. The strategies that have been used to design small molecules to target the interface antiparallel beta-sheet have produced lipopeptides, guanidinium derivatives and peptides (or peptidomimetics) cross-linked with spacers. The mechanism of inhibition was determined using a combination of kinetic and biophysical methods. These dimerization inhibitors proved equally active in vitro against both wild-type and mutated proteases. They are therefore promising alternatives to active-site-directed inhibitors in AIDS therapy. Disruption of protein-protein interactions by small molecules is a new way to obtain potentially therapeutic molecules.

Disruption of the HIV‐1 protease dimer with interface peptides: Structural studies using NMR spectroscopy combined with [2‐13C]‐Trp selective labeling

HIV-1 protease (HIV-1 PR), which is encoded by retroviruses, is required for the processing of gag and pol polyprotein precursors, hence it is essential for the production of infectious viral particles. In vitro inhibition of the enzyme results in the production of progeny virions that are immature and noninfectious, suggesting its potential as a therapeutic target for AIDS. Although a number of potent protease inhibitor drugs are now available, the onset of resistance to these agents due to mutations in HIV-1 PR has created an urgent need for new means of HIV-1 PR inhibition. Whereas enzymes are usually inactivated by blocking of the active site, the structure of dimeric HIV-1 PR allows an alternative inhibitory mechanism. Since the active site is formed by two half-enzymes, which are connected by a four-stranded antiparallel beta-sheet involving the N- and C- termini of both monomers, enzyme activity can be abolished by reagents targeting the dimer interface in a region relatively free of mutations would interfere with formation or stability of the functional HIV-1 PR dimer. This strategy has been explored by several groups who targeted the four-stranded antiparallel beta-sheet that contributes close to 75% of the dimerization energy. Interface peptides corresponding to native monomer N- or C-termini of several of their mimetics demonstrated, mainly on the basis of kinetic analyses, to act as dimerization inhibitors. However, to the best of our knowledge, neither X-ray crystallography nor NMR structural studies of the enzyme-inhibitor complex have been performed to date. In this article we report a structural study of the dimerization inhibition of HIV-1 PR by NMR using selective Trp side chain labeling.

Partially folded states of HIV-1 protease: Molecular dynamics simulations and ligand binding

All-atom molecular dynamics simulations were performed on partially folded states (with different secondary structure content) of the dimeric enzyme HIV-1 protease in aqueous solution. The calculations were based on previous simulations of the folding process of the protein based on a Go-model. The structures turn out to be stable, and the subunit–subunit contact surface is smaller than that of the native state. Interestingly, the flexibility of the partially folded states is similar to that observed for the monomer in the native state. The intersubunit contacts are formed by conserved residues, suggesting that these residues may play a role for the folding process. Docking a large set of molecules suggests that several ligands not yet associated to HIV-1 protease may bind to these partially unfolded structures.

Application of Novel Solid Lipid Nanoparticle (SLN)-Gene Vector Formulations Based on a Dimeric HIV-1 TAT-Peptide in Vitro and in Vivo

To optimize gene delivery of SLN-based gene vectors by incorporation of a dimeric HIV-1 TAT peptide (TAT2) into SLN gene vectors. Plasmid DNA was complexed with two SLN preparations either with or without pre-compaction of DNA by TAT2, poly-L-arginine, or the mutant TAT2-M1. DNA complexed with polyethylenimine (PEI) served as a standard. Gene expression was analyzed upon transfection of bronchial epithelial cells in vitro and after intratracheal instillation or aerosol application to the lungs of mice in vivo. Stability of DNA was analyzed by agarose gel electrophoresis. Incorporation of TAT2 into SLN gene vectors induced an up to 100-fold sequence-dependent increase of gene expression as compared with the mutant TAT2-M1 and was 4- to 8-times higher as compared with PEI in vitro. In vivo application of TAT2-SLN gene vectors via jet nebulization increased SLN-based gene expression but was accompanied with DNA degradation. DNA degradation was not observed when an innovative device operating on the principle of a perforated vibrating membrane was used. Incorporation of TAT2 into SLN gene vectors is suitable to optimize gene transfer in vitro. The use of a mild nebulization technology avoids DNA degradation and offers the opportunity for further studies in large animal models.

Simple, intuitive calculations of free energy of binding for protein-ligand complexes. 3. The free energy contribution of structural water molecules in HIV-1 protease complexes.

Structural water molecules within protein active sites are relevant for ligand-protein recognition because they modify the active site geometry and contribute to binding affinity. In this work an analysis of the interactions between 23 ligands and dimeric HIV-1 protease is reported. The X-ray structures of these complexes show the presence of four types of structural water molecules: water 301 (on the symmetry axis), water 313, water 313bis, and peripheral waters. Except for water 301, these are generally complemented with a symmetry-related set. The GRID program was used both for checking water locations and for placing water molecules that appear to be missing from the complexes due to crystallographic uncertainty. Hydropathic analysis of the energetic contributions using HINT indicates a significant improvement of the correlation between HINT scores and the experimentally determined binding constants when the appropriate bridging water molecules are taken into account. In the absence of water r2 = 0.30 with a standard error of +/- 1.30 kcal mol(-1) and when the energetic contributions of the constrained waters are included r2 = 0.61 with a standard error of +/- 0.98 kcal mol(-1). HINT was shown to be able to map quantitatively the contribution of individual structural waters to binding energy. The order of relevance for the various types of water is water 301 > water 313 > water 313bis > peripheral waters. Thus, to obtain the most reliable free energy predictions, the contributions of structural water molecules should be included. However, care must be taken to include the effects of water molecules that add information value and not just noise.

Role of the Trans-activation Response Element in Dimerization of HIV-1 RNA

The HIV-1 genome consists of two identical RNA strands that are linked together through non-covalent interactions. A major determinant for efficient dimerization of the two RNA strands is the interaction between palindromic sequences in the dimerization initiation site. Here we use an interplay of bioinformatics, biochemistry, and atomic force microscopy to describe another conserved palindrome in the trans-activation response element (TAR) that functions as a strong dimerization site when transiently exposed to the viral nucleocapsid protein. In conjunction with the DIS interaction, the TAR dimerization induces the formation of a 65-nm higher-order circular structure in the dimeric HIV-1 RNA. Our results provide a molecular model for the role of TAR in packaging and reverse transcription of the viral genome. The unique structure of the TAR-TAR dimer renders it an intriguing therapeutic target for the treatment of HIV-1 infection.

RNA Interactions in the 5′ Region of the HIV-1 Genome

The untranslated leader of the dimeric HIV-1 RNA genome is folded into a complex structure that plays multiple and essential roles in the viral replication cycle. Here, we have investigated secondary and tertiary structural elements within the 5′ 744 nucleotides of the HIV-1 genome using a combination of bioinformatics, enzymatic probing, native gel electrophoresis, and UV-crosslinking experiments. We used a recently developed RNA folding algorithm (Pfold) to predict the common secondary structure of an alignment of 20 divergent HIV-1 sequences. Combining this analysis with biochemical data, we present a secondary structure model for the entire 744 nucleotide fragment, which incorporates previously recognized and novel structural elements. In particular, our data provided strong evidence for a long-distance interaction between the region encompassing the AUG Gag initiation codon and an upstream region and we demonstrate that this feature is highly conserved in distantly related human and animal retroviruses. To obtain information about tertiary interactions we applied an intramolecular UV-crosslinking strategy and identified a novel tertiary interaction within the PBS hairpin structure.

Design and synthesis of dimeric HIV-1 integrase inhibitory peptides.

Dimers of known HIV-1 integrase inhibitory hexapeptide H-His-Cys-Lys-Phe-Trp-Trp-NH(2) containing different lengths of cross linkers in the place of cysteine residue, were designed, and synthesized. The inhibitory potency of these dimeric peptides is consistently higher than the lead hexapeptide. The dimeric peptide with djenkolic acid linker exhibited IC(50) values of 5.3 and 6.5 microM, for 3'-end processing and strand transfer, respectively.

Flexibility of Monomeric and Dimeric HIV-1 Protease

The flexibility and stability of both monomeric and dimeric HIV-1 PR were explored by 100 ns implicit solvent molecular dynamics simulation at 350 K with the aim to correlate the monomer stability with the dimerization mechanism. The principal component analysis (PCA) was applied to visualize the available regions in the conformational space of the two HIV-1 PR forms, to compare their structural diversity and to map the bottom of their underlying energy landscapes. It was found that whereas the flap tips (residues 45-55) are flexible and adopt close and open conformations in both monomeric and dimeric forms, the Nand C-termini (residues 1-4 and 96-99, respectively), which constitute the interface between the two subunits, are flexible only in the monomer. The different flexibility of the monomeric and dimeric HIV-1 PR is reflected in the different topography of their underlying energy landscape. Although the bottom of the monomer energy landscape is broad and rough, that of the dimer is narrower, deeper, and smoother, reflecting the enhanced flexibility of the monomer and the stabilizing interactions between the dimer subunits. Accordingly, blocking one or both terminals may prevent the formation of the active site. Despite the different flexibility of the termini in the monomeric and dimeric HIV-1 PR, their secondary structure contents are similar. The partial stability of the monomer together with the flexibility of its termini suggest that the HIV-1 PR is not a twostate dimer, as indicated by equilibrium denaturation experiments, but a three-state dimer with a marginally stable monomeric intermediate. This involves the swapping of the flexible termini across the two chains to form the dimer interface.

Interfacial peptide inhibitors of HIV-1 integrase activity and dimerization

Peptides derived from the interfacial region of dimeric HIV-1 integrase were evaluated as inhibitors of integrase's 3′-endonuclease activity. Three peptides were found to be moderately potent inhibitors with IC 50 values in the low micromolar range. The mode of inhibition was probed through protein crosslinking experiments. Active interfacial peptides were found to inhibit crosslinking of the dimeric form of integrase. Interfacial peptides that were poor inhibitors had no effect on integrase crosslinking.

Reverse transcriptase incorporation of 1,5-anhydrohexitol nucleotides.

Several reverse transcriptases were studied for their ability to accept anhydrohexitol triphosphates, having a conformationally restricted six-membered ring, as substrate for template-directed synthesis of HNA. It was found that AMV, M-MLV, M-MLV (H(-)), RAV2 and HIV-1 reverse transcriptases were able to recognise the anhydrohexitol triphosphate as substrate and to efficiently catalyse the incorporation of one non-natural anhydrohexitol nucleotide opposite a natural complementary nucleotide. However, only the dimeric enzymes, the RAV2 and HIV-1 reverse transcriptases, seemed to be able to further extend the primer with another anhydrohexitol building block. Subsequently, several HIV-1 mutants (4xAZT, 4xAZT/L100I, L74V, M184V and K65A) were likewise analysed, resulting in selection of K65A and, in particular, M184V as the most succesful mutant HIV-1 reverse transcriptases capable of elongating a DNA primer with several 1,5-anhydrohexitol adenines in an efficient way. Results of kinetic experiments in the presence of this enzyme revealed that incorporation of one anhydrohexitol nucleotide of adenine or thymine gave an increased (for 1,5-anhydrohexitol-ATP) and a slightly decreased (for 1,5-anhydrohexitol-TTP) K(m) value in comparison to that of their natural counterparts. However, no more than four analogues could be inserted under the experimental conditions required for selective incorporation. Investigation of incorporation of the altritol anhydrohexitol nucleotide of adenine in the presence of M184V and Vent (exo(-)) DNA polymerase proved that an adjacent hydroxyl group on C3 of 1,5-anhydrohexitol-ATP has a detrimental effect on the substrate activity of the six-ring analogue. These results could be rationalised based on the X-ray structure of HIV-1 reverse transcriptase.

Insertion of a peptide from MuLV RT into the connection subdomain of HIV-1 RT results in a functionally active chimeric enzyme in monomeric conformation.

The natural form of the human immunodeficiency virus type one reverse transcriptase (HIV-1 RT) found in virion particles is a heterodimer composed of the p66 and p51 subunits. The catalytic activity resides in the larger subunit in the heterodimeric (p66/p51) enzyme while in the monomeric form it is inactive. In contrast, Murine leukemia virus RT (MuLV RT) is functionally active in the monomeric form. In the primary amino acid sequence alignment of MuLV RT and HIV-1 RT, we have identified three specific regions in MuLV RT, that were missing in HIV-1 RT. In a separate study, we have shown that a chimeric RT construct comprising of the polymerase domain of HIV-1 RT and RNase-H domain of MuLV RT is functionally active as monomer [20]. In this communication, we demonstrate that insertion of a peptide (corresponding to amino acid residues 480-506) from the connection subdomain of MuLV RT into the connection subdomain of HIV-1 RT (between residues 429 and 430) results in a functionally active monomeric chimeric RT. Furthermore, this chimeric enzyme does not dimerize with exogenously added p51 subunit of HIV-1RT. Functional analysis of the chimeric RT revealed template specific variations in its catalytic activity. The chimeric enzyme catalyzes DNA synthesis on both heteropolymeric DNA and homopolymeric RNA (poly rA) template but curiously lacks reverse transcriptase ability on heteropolymeric RNA template. Similar to MuLV RT, the polymerase activity of the chimeric enzyme is not affected by acetonitrile, a reagent which dissociates dimeric HIV-1 RT into inactive monomers. These results together with a proposed 3-D molecular model of the chimeric enzyme suggests that the insertion of the missing region may induce a change in the spatial position of RNase H domain such that it is functionally active in monomeric conformation.

Bioanalysis of syn dimeric HIV-1 protease inhibitor N-benzyl 4-aryl-1,4-dihydropyridine H19: metabolic and cytotoxic properties in Hep G2 cells.

Syn dimeric N-benzyl 4-aryl-1,4-dihydropyridine H19 is a nonpeptidic HIV-1 protease inhibitor of the dihydroxyethylene type representing novel C2-symmetric inhibitors. Great interest was focussed on the extent of metabolism of these novel inhibitory structures as their functional groups are similar to certain peptidic and non-peptidic HIV-1 protease inhibitors with poor bioavailability due to extensive metabolism. Thus, early characterization of metabolic and toxic properties decisively determines the future prospects of those novel HIV-1 protease inhibitors. Both metabolism and toxicity were evaluated in Hep G2 monolayers. While no phase-I metabolites were found the extent of conjugation in phase-II of biotransformation was poor. Moreover, cytotoxic evaluation of protein and DNA decrease and, furthermore, of membrane toxicity characterized the novel inhibitors as non-toxic. Consequently, the favourable poor metabolism and non-toxic properties encourage further development of these novel HIV-1 protease inhibitors.

The HIV-1(Lai) RNA dimerization. Thermodynamic parameters associated with the transition from the kissing complex to the extended dimer.

Retroviruses contain dimeric RNA consisting of two identical copies of the genomic RNA. The interaction between these two RNA molecules occurs near their 5' ends. A region upstream from the splice donor comprising an auto-complementary sequence has been identified as being responsible for the initiation of the formation of dimeric HIV-1(Lai) RNA. This region (SL1), part of the PSI encapsidation domain, can adopt a stem-loop structure. It has already been shown that this stem-loop structure can initiate the formation of two distinct dimers differing in their thermostability: a loop-loop dimer or 'kissing complex' and an extended dimer. We report here a study using UV and 1D NMR spectroscopy of the dimerization of a short oligoribonucleotide (23 nucleotides) spanning nucleotides 248-270 of the HIV-1(Lai) SL1 sequence, in order to derive the thermodynamic parameters associated with the transition from the loop-loop complex to the extended dimer. The temperature dependence of the UV absorbency shows an hypochromicity for this transition with a small enthalpy change equal to - 29.4 +/- 5 kcal x mol-1, together with a concentration independent transition which implies a monomolecular reaction. On the other hand, our NMR results don't indicate a dissociation of the GCGCGC sequence engaged in the loop-loop interaction during the rearrangement of the loop-loop complex into the extended dimer. Our data suggest that the loop-loop interaction is maintained during the temperature dependent conformational change while the intramolecular base-pairing of the stems is disrupted and then reconstituted to form an intermolecular base-pairing leading to an extended dimer.

Human immunodeficiency virus type 1 reverse transcriptase dimer destabilization by 1-[Spiro[4"-amino-2",2" -dioxo-1",2" -oxathiole-5",3'-[2', 5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]]]-3-ethylthy mine.

The nonnucleoside inhibitor binding pocket is a well-defined region in the p66 palm domain of the human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT). This binding pocket opens toward the interface of the p66/p51 heterodimer and we have investigated whether ligand binding at or near this site induces structural changes that have an impact on the dimeric structure of HIV-1 RT. 1-[2',5'-bis-O-(tert-butyldimethylsilyl]-3'-spiro-5' '-(4' '-amino-1' ',2' '-oxathiole-2' ',2' '-dioxide)-3-ethylthymine (TSAOe(3)T) was found to destabilize the subunit interactions of both the p66/p51 heterodimer and p66/p66 homodimer enzymes. The Gibbs free energy of dimer dissociation (DeltaG(D)(H)2(O)) is decreased with increasing concentrations of TSAOe(3)T, resulting in a loss in dimer stability of 4.0 and 3.2 kcal/mol for the p66/p51 and p66/p66 HIV-1 RT enzymes, respectively. This loss of energy is not sufficient to induce the dissociation of the subunits in the absence of denaturant. This destabilizing effect seems to be unique for TSAOe(3)T, since neither the tight-binding inhibitor UC781 nor nevirapine showed any effects on the stability of HIV-1 RT dimers. TSAOe(3)T was unable to destabilize the subunit interactions of the E138K mutant enzyme, which exhibits significant resistance to TSAOe(3)T inhibition. Molecular modeling of TSAOm(3)T into the nonnucleoside inhibitor binding pocket of wild-type RT suggests that it makes significant interactions with the p51 subunit of the enzyme, a feature that has not been observed with other types of nonnucleoside inhibitors. The observed destabilization of the dimeric HIV-1 RT may result from structural/conformational perturbations at the reverse transcriptase subunit interface.

First bioanalytical evaluation of nonpeptidic cage dimeric HIV-1 protease inhibitor N-benzyl 4-aryl-1,4-dihydropyridine H17: biotransformation and toxicity on Hep G2 cells.

Cage dimeric N-benzyl 4-aryl-1,4-dihydropyridine H17 is a moderate inhibitor of HIV-1 protease. As representative of an innovative and promising class of nonpeptidic HIV-1 protease inhibitors H17 was selected for the characterization of the biochemical profile of the cage dimers concerning metabolic and toxic aspects. In the first bioanalytical evaluation of H17 on Hep G2 monolayers no phase-I metabolites were found and the extent of conjugation on phase-II of biotransformation was poor due to steric hindrance of the hydroxymethylene groups. H17 was found to be nearly non-toxic. A slight noticeable influence on cell proliferation, however, did not result from apoptotic activities. Thus, first biochemical evaluation of H17 practically suggests no decrease of an in-vivo bioavailability by metabolization.