TAR RNA binding properties and relative transactivation activities of human immunodeficiency virus type 1 and 2 Tat proteins.

Using gel shift assays, we found that the human immunodeficiency virus type 1 (HIV-1) Tat protein (Tat-1) bound both HIV-1 and HIV-2 TAR RNAs with similar high affinities. In contrast, the HIV-2 Tat protein (Tat-2) bound only TAR-2 RNA with high affinity. We conclude that the weak in vivo activity of Tat-2 on the HIV-1 long terminal repeat that has been observed previously is likely the result of low affinity for TAR-1 RNA. Additionally, TAR-2 RNA was found to contain multiple specific binding sites for Tat proteins. GAL4-Tat fusion proteins were analyzed to compare the relative transactivation activities of Tat-1 and Tat-2 in the absence of requirements for binding to TAR RNAs. The GAL4-Tat-2 protein was found to transactivate synthetic promoters containing GAL4 binding sites at levels severalfold higher than did the GAL4-Tat-1 protein.     

The basic RNA-binding domain of HIV-2 Tat contributes to preferential trans-activation of a TAR2-containing LTR.

Human immunodeficiency viruses HIV-1 and HIV-2 encode a Tat protein that trans-activates the respective viral genome through RNA targets (TAR1 and TAR2). Tat-1 and Tat-2 have considerable homology. However, an interesting biological observation has been that Tat-1 activates the HIV-1 and HIV-2 LTRs equally while Tat-2 activates the former, in comparison to the latter, poorly. Here, we present evidence that it is the TAR2 RNA target together with the basic RNA-binding protein domain of Tat-2 that dictate this non-reciprocity in trans-activation.     

HIV-1 Tat directly binds to NFkappaB enhancer sequence: role in viral and cellular gene expression

HIV-1 Tat protein reprograms cellular gene expression of infected as well as uninfected cells apart from its primary function of transactivating HIV-1 long terminal repeat (LTR) promoter by binding to a nascent RNA stem–loop structure known as the transactivator response region (TAR). Tat also induces chromatin remodeling of proviral LTR-mediated gene expression by recruiting histone acetyl transferases to the chromatin, which results in histone acetylation. Furthermore several studies have shown convincing evidence that Tat can transactivate HIV-1 gene expression in the absence of TAR, the molecular mechanism of which remains to be elucidated. Here we show a direct interaction of Tat with nuclear factor kappa B (NFκB) enhancer, a global regulatory sequence for many cellular genes both in vitro and in vivo. This interaction not only provides a novel molecular basis to explain TAR-independent transactivation in HIV-1, but also points toward the potential mechanism of Tat- mediated modulation of cellular genes.     

Oligonucleotide inhibition of the interaction of HIV-1 Tat protein with the trans-activation responsive region (TAR) of HIV RNA

The interaction of HIV-1 Tat protein with its recognition sequence, the trans-activation responsive region TAR is a potential target for drug discovery against HIV infection. We show by use of an in vitro competition filter binding interference assay that synthetic oligodeoxyribonucleotides complementary to the HIV-1 TAR RNA apical stem-loop and bulge region inhibit the binding of Tat protein or a Tat peptide (residues 37-72) better than two small molecules that have been shown to bind TAR RNA, Hoechst 33258 and neomycin B. The inhibition is not sensitive to length between 13 and 16 residues or precise positioning but shorter oligonucleotides are less effective. Enhanced inhibition was obtained for a 16-mer 2'-O-methyl oligoribonucleotide but not for C5-propyne pyrimidine-substituted oligonucleotides. Control non-antisense oligonucleotides were occasionally also effective in filter binding interference but only the complementary antisense 2'-O-methyl oligoribonucleotide was effective in gel mobility shift assays in direct TAR binding or in interference with Tat peptide binding to the TAR stem-loop. This is the first demonstration of effective inhibition of the Tat-TAR interaction by nuclease-stabilized oligonucleotide analogues.     

Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3

B7-H3 (CD276) is both an inhibitory ligand for natural killer cells and T cells and a tumor antigen that is widely expressed among human solid tumors. Anti-B7-H3 mouse monoclonal antibody 8H9 has been successfully used for radioimmunotherapy for patients with B7-H3(+) tumors. We present the humanization, affinity maturation, and epitope mapping of 8H9 based on structure determination, modeling, and yeast display methods. The crystal structure of ch8H9 Fab fragment was solved to 2.5-Å resolution and used as a template for humanization. By displaying the humanized 8H9 single chain Fv (scFv) on the surface of yeast, the affinity was matured by sequential random mutagenesis and fluorescence-activated cell sorting. Six mutations (three in the complementarity-determining region and three in the framework regions) were identified and incorporated into an affinity-matured humanized 8H9 construct (hu8H9-6m) and an affinity-matured chimeric 8H9 construct (ch8H9-6m). The hu8H9-6m scFv had a 160-fold improvement in affinity (0.9 nm K_D) compared with parental hu8H9 scFv (144 nm K_D). The IgG formats of ch8H9-6m and hu8H9-6m (nanomolar to subnanomolar K_D) had 2–9-fold enhancements in affinity compared with their parental forms, potent in vitro antibody-dependent cell-mediated cytotoxicity (0.1–0.3 μg/ml EC₅₀), and high tumor uptake in mouse xenografts. Based on in silico docking studies and experimental validation, the molecular epitope of 8H9 was determined to be dependent on the FG loop of B7-H3, a region critical to its function in immunologic blockade and unique among anti-B7-H3 antibodies published to date.     

Deciphering structure-activity relationships in a series of Tat/TAR inhibitors

A series of pentameric “Polyamide Amino Acids” (PAAs) compounds derived from the same trimeric precursor have been synthesized and investigated as HIV TAR RNA ligands, in the absence and in the presence of a Tat fragment. All PAAs bind TAR with similar sub-micromolar affinities but their ability to compete efficiently with the Tat fragment strongly differs, IC₅₀ ranging from 35 nM to >2 μM. While NMR and CD studies reveal that all PAA interact with TAR at the same site and induce globally the same RNA conformational change upon binding, a comparative thermodynamic study of PAA/TAR equilibria highlights distinct TAR binding modes for Tat competitor and non-competitor PAAs. This led us to suggest two distinct interaction modes that have been further validated by molecular modeling studies. While the binding of Tat competitor PAAs induces a contraction at the TAR bulge region, the binding of non-competitor ones widens it. This could account for the distinct PAA ability to compete with Tat fragment. Our work illustrates how comparative thermodynamic studies of a series of RNA ligands of same chemical family are of value for understanding their binding modes and for rationalizing structure-activity relationships.     

Cloning,expression, and identification of anti‐carbofuran single chain Fv gene

Phage display method was used to clone anti‐carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF‐specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly₄Ser)₃. The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti‐CBF antibody libraries were then constructed. After one round of panning with CBF‐ovalbumin (CBF‐OVA) as a conjugate, antigen‐binding positive recombinant phage clones were successfully selected by enzyme‐linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS‐PAGE and showed HRP‐anti‐E‐tag antibody‐recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC₅₀ value of 1.07 ng/mL. The cross reactivity studies showed that the anti‐CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Kinetic characterization of TAR RNA-Tat peptide and neomycin interactions by acoustic wave biosensor

The kinetics of binding of short Tat peptides and an aminoglycoside molecule to the human immunodeficiency virus-type 1(HIV-1) TAR RNA and to a bulge mutant analogue (MTAR) is studied in a biosensor format by monitoring the time course of the response in a series resonance frequency, using an acoustic wave biosensor. Association and dissociation rate constants are evaluated by fitting the experimental data to a simple 1:1 (Langmuir) model. Kinetic rate and equilibrium dissociation constants show that MTAR-peptide complexes are subject to a higher dissociation rate and are less stable compared to the corresponding TAR-peptide complexes. In addition, longer peptides display enhanced discrimination ability than a shorter peptide according to the equilibrium dissociation constants evaluated using this technique. K(D) values for TAR-Tat vs. MTAR-Tat complexes are 2.6 vs. 3.8 microM for Tat-12, 0.87 vs. 4.3 microM for Tat-18 and 0.93 vs. 1.6 microM for Tat-20. The equilibrium dissociation constant for TAR-neomycin complex is 12.4 microM and it is comparable to the values obtained from non-biosensor type assays. These findings are in parallel with those cited in the literature and the results from this study underline the potential of the acoustic wave sensor for detailed biophysical analysis of nucleic acid-ligand binding.     

Cloning of a single-chain variable fragment (scFv) switching active plasminogen activator inhibitor-1 to substrate

Increased levels of plasminogen activator inhibitor-1 (PAI-1) are a well-known risk for cardiovascular diseases. A significant number of investigations are aimed at lowering plasma levels of PAI-1 to enhance endogenous fibrinolysis. We have recently generated monoclonal antibodies that neutralize PAI-1 activity by switching the inhibitory conformation to a substrate conformation. However, intact murine antibodies have quite some disadvantages for therapeutic use in man. In the current study, we describe the construction of a smaller antibody fragment derived from a monoclonal antibody (MA-8H9D4) with PAI-1 neutralizing properties. The cDNAs encoding the variable domains of the heavy and light chain were amplified, linked and cloned into a phagemid vector. Resulting clones were expressed as a single-chain variable fragment (scFv, V_H-(Gly₄Ser)₃-V_L) on the surface of a phage and selected for binding to PAI-1. Subsequently, a positive phage was used for the production of soluble scFv-8H9D4. Following purification, the characteristics of the scFv-8H9D4 were compared to those of the original MA-8H9D4. The scFv inhibited PAI-1 activity to a similar extent as MA-8H9D4 and by a similar mechanism, i.e., induction of a conformational switch. Thus, this smaller antibody fragment, exhibiting the same properties as the parent molecule may constitute a useful starting point for the design of PAI-1 neutralizing therapeutics. © 1997 Elsevier Science B.V. All rights reserved.