Inhibition of HIV-1 proteinase by metal ions.

1. Certain metal ions have been identified as inhibitors (IC50 1-20 microM) of the aspartic proteinase of Human Immunodeficiency Virus Type 1 (HIV-PR). 2. By contrast most simple metal ions do not inhibit this enzyme. 3. Those that did inhibit have in common a high charge/size ratio or "hard" acidic nature, preferring to combine covalently with oxygen donor ligands. 4. Some evidence from independent X-ray crystal structure determinations suggests that the metalloinhibitors identified here may bind in the active site of the enzyme via coordination to the carboxylate side chains of the essential active site residues Asp 25 and 125. 5. Although the measured inhibition is only microM, very few enzyme-inhibitor interactions can be taking place and so more complex metalloinhibitors with ligands that can also bind to peptide side chains of the enzyme might be significantly more potent inhibitors of HIV-PR and of viral replication.     

Inhibition of the aspartic proteinase from HIV-2

Kinetic constants were determined for the interaction of the HIV-2 aspartic proteinase with a synthetic substrate and a number of inhibitors at several pH values. Acetyl-pepstatin was more effective towards HIV-2 proteinase than the renin inhibitor, H-261; this effect is exactly the opposite from that observed previously for the proteinase from the HIV-1 AIDS virus.     

Flavones are inhibitors of HIV-1 proteinase.

Substituted gamma-chromones were found to weakly inhibit HIV-1 proteinase, an important enzyme in the replication and processing of the AIDS virus. Chromones bearing hydroxyl substituents and a phenolic group at the 2-position (flavones) were the most active compounds and structure-activity relationships for a limited series of flavone inhibitors are presented. Dixon plots are reported and a possible mechanism for flavone-induced inhibition is proposed. The results are also compared with those for some structurally related non-peptidic inhibitors of HIV-1 proteinase. Since some flavonoid compounds have already been shown to have antiviral activity against AIDS, the present observations of anti-HIV-1 proteinase activity may be particularly significant.     

Non-peptidic anti-AIDS agents: inhibition of HIV-1 proteinase by disulfonates.

Based upon an earlier observation that sodium docosanedioate (NaO2C-(CH2)20-CO2NA) weakly inhibits HIV-1 proteinase (IC50 12 microM), we have identified a class of more potent inhibitors (sulfonic acids) of this enzyme which are likewise dianionic at pH 5-6.5. Many of the compounds were moderately strong inhibitors of the enzyme (IC50 40nM-10 microM) and some have previously been shown to have anti-HIV activity in lymphocytes. Proteinase inhibition was dependent on the separation between sulfonate/carboxylate substituents, consistent with the hypothesis that negative charged ends of an inhibitor might form ionic bonds with Arg 8 and Arg 108 located at either end of the substrate-binding groove of the enzyme. The binding mode remains to be established by structure elucidation. Results for enzyme inhibition are presented along with structure-activity relationships and evidence for pH dependent inhibition. The general observations reported here may be useful for developing more potent and selective non-peptidic proteinase inhibitors.     

Inhibition of the HIV-1 and HIV-2 proteases by a monoclonal antibody

The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates. This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity.     

Conformational changes in HIV-1 proteinase: effect of protonation of the active center on conformation of HIV-1 proteinase in water

At weak acidic pH, where HIV-1 proteinase is most stable and active, its catalytic Asp 25/25' dyad shares one proton. At a physiological pH the dyad is deprotonated, however, 2 ns molecular dynamics simulations of the HIV-1 protease with monoprotonated and deprotonated Asp25/25' dyad is performed, in order to investigate the influence of Asp25/25' protonation state on the proteinase dynamics. For net charge neutralization the 4 Cl- ions were included. In case of deprotonated active site the significant tertiary structure deviation of HIV-1 PR structure from crystal structure is observed, while in the monoprotonated one the tertiary structure fluctuates near starting structure. Possible mechanism of the influence of the Asp25/25' protonation state on proteinase dynamics is discussed.     

Efficient Gene Transfer Mediated by HIV-1-based Defective Lentivector and Inhibition of HIV-1 Replication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5-1.2 × 107IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols.     

A Computational Model of Inhibition of HIV-1 by Interferon-Alpha

Type 1 interferons such as interferon-alpha (IFNα) inhibit replication of Human immunodeficiency virus (HIV-1) by upregulating the expression of genes that interfere with specific steps in the viral life cycle. This pathway thus represents a potential target for immune-based therapies that can alter the dynamics of host-virus interactions to benefit the host. To obtain a deeper mechanistic understanding of how IFNα impacts spreading HIV-1 infection, we modeled the interaction of HIV-1 with CD4 T cells and IFNα as a dynamical system. This model was then tested using experimental data from a cell culture model of spreading HIV-1 infection. We found that a model in which IFNα induces reversible cellular states that block both early and late stages of HIV-1 infection, combined with a saturating rate of conversion to these states, was able to successfully fit the experimental dataset. Sensitivity analysis showed that the potency of inhibition by IFNα was particularly dependent on specific network parameters and rate constants. This model will be useful for designing new therapies targeting the IFNα network in HIV-1-infected individuals, as well as potentially serving as a template for understanding the interaction of IFNα with other viruses.     

Inhibition of HIV-1 replication by cell-penetrating peptides binding Rev

New therapeutic agents able to block HIV-1 replication are eagerly sought after to increase the possibilities of treatment of resistant viral strains. In this report, we describe a rational strategy to identify small peptide sequences owning the dual property of penetrating within lymphocytes and of binding to a protein target. Such sequences were identified for two important HIV-1 regulatory proteins, Tat and Rev. Their association to a stabilizing domain consisting of human small ubiquitin-related modifier-1 (SUMO-1) allowed the generation of small proteins named SUMO-1 heptapeptide protein transduction domain for binding Tat (SHPT) and SUMO-1 heptapeptide protein transduction domain for binding Rev (SHPR), which are stable and efficiently penetrate within primary lymphocytes. Analysis of the antiviral activity of these proteins showed that one SHPR is active in both primary lymphocytes and macrophages, whereas one SHPT is active only in the latter cells. These proteins may represent prototypes of new therapeutic agents targeting the crucial functions exerted by both viral regulatory factors.     

Efficient Gene Transfer Mediated by HIV-1-based Defective Lentivector and Inhibition of HIV-1 Repfication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 （HIV-1）-based defective lentiviral vectors （DLV） and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV （0.5-1.2 × 107IU/mL）, which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols.     

Sensitive, soluble chromogenic substrates for HIV-1 proteinase

By replacement of the P1' residue in a capsid/nucleocapsid cleavage site mimic with 4-NO2-phenylalanine (Nph), an excellent chromogenic substrate, Lys-Ala-Arg-Val-Leu*Nph-Glu-Ala-Met, for HIV-1 proteinase (kappa cat = 20 s-1, Km = 22 microM) has been prepared. Substitution of the Leu residue in P1 with norleucine, Met, Phe, or Tyr had minimal effects on the kinetic parameters (kappa cat and kappa cat/Km) determined at different pH values, whereas peptides containing Ile or Val in P1 were hydrolyzed extremely slowly. The spectrophotometric assay has been used to characterize the proteinase further with respect to pH dependence, ionic strength dependence, and the effect of competitive inhibitors of various types.     

Intrinsic activity of precursor forms of HIV-1 proteinase.

The wild-type -Phe*Pro- bond located at the N-terminus of the mature aspartic proteinase of HIV-1 was replaced by -Ile-Pro- or -Val-Pro-. By this means, processing at this cleavage junction was prevented and so, extended or precursor forms of HIV-proteinase were generated. These constructs were expressed in Escherichia coli, purified therefrom, and their specificity, activity at different pH values and susceptibility to the potent inhibitor, Ro31-8959, was assessed. A hitherto unobserved cleavage junction (at approximately Ala-Phe*Leu-Gln approximately) in the frame-shift region of the gag-pol viral genome was identified and confirmed by demonstrating cleavage of a synthetic peptide corresponding to this region. The implications for viral replication of self-processing at neural pH by proteinase whilst still present (in a precursor form) as a component of the polyprotein are considered; such reactions, however, are still blocked even at pH values as high as 8.0 by Ro31-8959.     

Synthesis of specific peptide substrates for HIV-1 proteinase.

Two small peptide substrates for HIV-1 proteinase were synthesised. The sequences chosen were basically from that of the gag-pol protein, which is the natural substrate for the proteinase. To protect these peptides from the attack of exopeptidases, the N- and C-termini were suitably protected, which also makes these substrates specific to HIV-proteinase and eliminates the requirement for highly purified enzyme.     

Inhibition of HIV-1 Tat-mediated LTR transactivation and HIV-1 infection by anti-Tat single chain intrabodies.

Genes encoding the rearranged immunoglobulin heavy and light chain variable regions of anti-HIV-1 Tat, exon 1 or exon 2 specific monoclonal antibodies have been used to construct single chain intracellular antibodies 'intrabodies' for expression in the cytoplasm of mammalian cells. These anti-Tat single chain intrabodies (anti-Tat sFvs) are additionally modified with a C-terminal human C kappa domain to increase cytoplasmic stability and/or the C-terminal SV40 nuclear localization signal to direct the nascent intrabody to the nuclear compartment, respectively. The anti-Tat sFvs with specific binding activity against the N-terminal activation domain of Tat, block Tat-mediated transactivation of HIV-1 LTR as well as intracellular trafficking of Tat in mammalian cells. As a result, the transformed lymphocytes expressing anti-Tat sFvs are resistant to HIV-1 infection. Thus, these studies demonstrate that stably expressed single chain intrabodies and their modified forms can effectively target molecules in the cytoplasm and nuclear compartments of eukaryotic cells. Furthermore, these studies suggest that anti-Tat sFvs used either alone or in combination with other genetically based strategies may be useful for the gene therapy of HIV-1 infection and AIDS.