No abstract

HIV-1 transactivating protein Tat is essential for virus replication and progression of HIV disease. HIV-1 Tat stimulates transactivation by binding to HIV-1 transactivator responsive element (TAR) RNA, and while secreted extracellularly, it acts as an immunosuppressor, an activator of quiescent T-cells for productive HIV-1 infection, and by binding to CXC chemokine receptor type 4 (CXCR4) as a chemokine analogue. Here we present a novel HIV-1 Tat antagonist, a neomycin B-hexaarginine conjugate (NeoR), which inhibits Tat transactivation and antagonizes Tat extracellular activities, such as increased viral production, induction of CXCR4 expression, suppression of CD3-activated proliferation of lymphocytes, and upregulation of the CD8 receptor. Moreover, Tat inhibits binding of fluoresceine isothiocyanate (FITC)-labeled NeoR to human peripheral blood mononuclear cells (PBMC), indicating that Tat and NeoR bind to the same cellular target. This is further substantiated by the finding that NeoR competes with the binding of monoclonal Abs to CXCR4. Furthermore, NeoR suppresses HIV-1 binding to cells. Importantly, NeoR accumulates in the cell nuclei and inhibits the replication of M- and T-tropic HIV-1 laboratory isolates (EC(50) = 0.8-5.3 microM). A putative model structure for the TAR-NeoR complex, which complies with available experimental data, is presented. We conclude that NeoR is a multitarget HIV-1 inhibitor; the structure, and molecular modeling and dynamics, suggest its binding to TAR RNA. NeoR inhibits HIV-1 binding to cells, partially by blocking the CXCR4 HIV-1 coreceptor, and it antagonizes Tat functions. NeoR is therefore an attractive lead compound, capable of interfering with different stages of HIV infection and AIDS pathogenesis.

Resonance Raman spectra of myoglobins reconstituted with hemes isotopically substituted at the central iron atom or the pyrrole nitrogen atoms have been recorded to address the issue of whether the strong line at ~220 cm"1 is the iron-histidine stretching mode or the iron-pyrrole nitrogen stretching mode. The frequency of the line at 220 cm'1 is 1.7 cm"1 lower in myoglobin reconstituted with the 57Fe heme than it is in the 54Fe-substituted heme. No large shifts were detected in any other Raman lines. When myoglobin reconstituted with 15N-substituted pyrrole nitrogens in the heme is compared to the unsubstituted myoglobin no large change is detected in the line at 220 cm"1, but the frequency of the line at 243 cm"1 is 1.5 cm"1 lower. In comparing myoglobin buffered in D20 to that buffered in H20 only the line at 220 cm"1 changes frequency (1.4 cm"1). From these isotopic substitution studies, we conclude that the line at ~220 cm"1 in myoglobin is the iron-histidine stretching mode. The mode at ~243 cm"1 has a significant contribution from the pyrrole nitrogens, and it is likely an out-of-plane pyrrole tilting mode. The 54Fe-57Fe isotope shift of 1.7 cm"1 in the 220-cm"1 line is smaller than predicted for a diatom oscillator of the iron and the histidine. We conclude that the iron-histidine stretching mode is either mixed with an internal mode of the histidine and/or mixed with skeletal modes of the porphyrin macrocycle.Resonance Raman scattering has been applied extensively to 7 AT&T Bell Laboratories.

No abstract

To study the meclhanism of arrest of DNA synthesis at d(TC ,ug of DNA, before addition of the polymerase. These assays were performed at pH 7.5.Synthesis of Single-Stranded DNA Containing 7-deaza A or 7-deaza G. DNA strands containing 7-deaza A were synthesized by a polymerase chain reaction using single-stranded mpTC27 phage DNA (at 2.5 ,ug/ml) as a template and the M13 17-mer (at 2.5 ug/ml) as a (single) primer. The reaction mixtures also contained 300 ,uM (each) 7-deaza dATP, dCTP, dGTP, and dTTP, 50 units of the Taq DNA polymerase (Thermus aquaticus DNA polymerase) per ml (New England Abbreviation: SSB, single-strand binding protein.

Regulation of HIV gene expression is crucially dependent on binding of the trans-activator protein, Tat, to the trans-activation response RNA element, TAR, found at the 5′ end of all HIV-1 transcripts. Tat-TAR interaction is mediated by a short arginine-rich domain of the protein. Disruption of this interaction could, in theory, create a state of complete viral latency. A new class of small-molecule peptidomimetic TAR RNA binders, conjugates of aminoglycosides and arginine, was recently designed [Litovchick, A., Evdokimov, A. G., and Lapidot, A. (1999) FEBS Lett. 445, 73-79]. Two of these compounds, the tri-arginine derivative of gentamicin C (R3G) and the tetra-arginine derivative of kanamycin A (R4K), bind efficiently and specifically to TAR RNA. These compounds display negligible toxicity while being transported and accumulated in cell nuclei. Here we present a detailed synthesis and chemical characterization of the aminoglycoside-arginine conjugates R3G and R4K as well as GB4K, the tetra-γ-guanidinobutyric derivative of kanamycin A. Their binding sites on TAR RNA were assigned by RNase A, uranyl nitrate, and lead acetate footprinting. The conjugates interact with TAR RNA in the widened major groove, formed by the UCU bulge and the neighboring base pairs of the upper stem portion of TAR, the binding site of Tat protein, and Tat-derived peptides (e.g., R52). Our results suggest an additional binding site of R4K and R3G compounds, in the lower stem-bulge region of TAR. The antiviral activity of the conjugates in cultured equine dermal fibroblasts infected with equine infectious anemia virus, used as a model system of HIV-infected cells, is also presented.The trans-activation responsive RNA (TAR) 1 region of HIV long terminal repeat (LTR) regulates the viral gene expression via interaction with the HIV trans-activator protein, Tat, and thus is an attractive target for drug design strategies (1). TAR is found at the 5′ end of all HIV-1 transcripts. It adopts a hairpin secondary structure consisting of a highly conservative hexanucleotide loop and a threenucleotide bulge flanked by two double-stranded stems (2). TAR is a positive enhancer that stimulates the synthesis of productive transcripts. It is unique in terms of eukaryotic transcription control because it only functions as an RNA element. The activation by Tat is entirely dependent on the presence of the TAR RNA sequence. Tat activates expression by specific binding to TAR, which increases viral mRNA production several hundredfold by stimulation of the elongation capacity of RNA polymerase II (3). HIV Tat binds the cyclin T subunit of P-TEFb and recruits P-TEFb to the HIV-1 LTR promoter. This process requires binding of Tat to the TAR bulge and of cyclin T to the TAR loop. The cyclin T-associated CDK9 kinase then induces phosphorylation of the C-terminal domain of RNA polymerase II, and of other polymerase II-associated proteins, leading to the transition from nonprocessive to processive transcription (4).Binding of Tat protein to TAR is mediated ...

The major cellulose-binding domain (CBD) from the cellulosome of Clostridium thermocellum YS was cloned and overexpressed in Escherichia coli. The expressed protein was purified efficiently by a modification of a novel procedure termed affinity digestion. The properties of the purified polypeptide were compared with those of a related CBD derived from a cellulosome-like complex of a similar (but mesophilic) clostridial species, Clostridium cellulovorans. The binding properties of the two proteins with their common substrate were found to be very similar. Despite the similarity in the amino acid sequences of the two CBDs, polyclonal antibodies raised against the CBD from C. thermocellum failed to interact with the protein from C. cellulovorans. Chemical modification of the single cysteine of the CBD had little effect on the binding to cellulose. Biotinylation of this cysteine allowed the efficient binding of avidin to cellulose, and the resultant matrix is appropriate for use as a universal affinity system.

HIV gene expression is crucially dependent on binding of the viral Tat protein to the transactivation RNA response element. A number of synthetic Tat-transactivation responsive element interaction inhibitors of peptide/peptoid nature were described as potential antiviral drug prototypes. We present a new class of peptidomimetic inhibitors, conjugates of L-arginine with aminoglycosides. Using a gel-shift assay and affinity chromatography on an L-arginine column we found that these compounds bind specifically to the transactivation responsive element RNA in vitro with K d values in the range of 20^400 nM, which is comparable to the K d of native Tat bound to the transactivation responsive element (10^12 nM). Confocal microscopy studies demonstrated that fluorescein-labelled conjugate penetrates into live cells. High affinity to the transactivation responsive element, low toxicity, and relative simplicity of synthesis make these compounds attractive candidates for antiviral drug design.z 1999 Federation of European Biochemical Societies.