Role of V3 independent domains on a dualtropic human immunodeficiency virus type 1 (HIV-1) envelope gp120 in CCR5 coreceptor utilization and viral infectivity

The molecular mechanism of human immunodeficiency virus type 1 (HIV-1) entry into cells involves specific interactions between the viral envelope glycoprotein gp120 and two target cell proteins, CD4 and either CCR5 or CXCR4 chemokine receptors. In order to delineate the functional role of HIV-1 gp120 subdomains of dualtropic strains in CCR5 coreceptor usage, we used a panel of chimeric viruses in which the V1/V2 and V3 domains of gp120 from the dualtropic HIV-1(KMT) isolate were introduced either alone or in combination into the T-tropic HIV-1(NL4-3) background. These chimeric constructs were employed in cell-cell fusion and cell-free virus infectivity assays using cell lines expressing CD4 and the CCR5 chemokine receptor. In both assays, the V3 domain of HIV-1(KMT) but not the V1/V2 domain proved to be the principal determinant of CCR5 coreceptor usage. However, in the cell-free viral infectivity assay although a chimeric virus with a combined V1/V2 and V3 domains of HIV-1(KMT) efficiently fused with coreceptor expressing cells, yet its infectivity was markedly diminished in CCR5 as well as CXCR4 expressing cells. Restoring a comparable level of infection of such chimeric virus required the C3-V5 domain from HIV-1(KMT) to be introduced. Our present findings confirmed that the V3 domain is the major determinant of fusion activity and cellular tropism, and demonstrated a dispensable role for the V1/V2 domain. In addition the C3-V5 domain appeared to play an important role in viral infectivity when the corresponding V1/V2 and V3 domains are present.     

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.     

Enhancement of Human Immunodeficiency Virus Type 1 (HIV-1) Infection via Increased Membrane Fluidity by a Cationic Polymer

Cationic polymers are known to have potent activity against bacteria, but their effects on viral activity have been little studied. We investigated the effect of one such polymer, polyethyleneimine (PEI), on HIV-1 infection. Although virus-cell binding was significantly inhibited by PEI, HIV-1 infection in human T-cell lines such as MT-4 and MOLT-4 was accelerated conversely when the drug treatment was carried out, after the virus had attached to the cells or PEI was simultaneously added to the virus and cell culture system. This paradoxical effect of PEI on HIV-1 infection was examined using HIV-1 chronically infected cells (MOLT-4/HIV-1). Dissociation of the glycoprotein gp120 (as revealed by exposure of transmembrane protein gp41) from MOLT-4/HIV-1 cells and the resultant fusion of these cells was shown to be induced by the addition of PEI. Accordingly, it was suggested that the binding inhibition of HIV-1 to CD4-positive cells by PEI was due to the shedding of gp120 from HIV-1 particles, and this PEI rather promoted membrane fusion between the virus and cells leading to the enhancement of HIV-1 infection. Similarly, dissociation of gp120 from MOLT-4/HIV-1 was also induced by sCD4. The effect of these reagents on changes in membrane fluidity was evaluated by polarization (p) measurements, and it was observed that the acceleration of membrane fluidity occurred only in the PEI system. Therefore, it is likely that PEI accelerates HIV-1 infection by facilitating virus entry into the host cells through an increase in membrane fluidity.     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.     

Suppression of HIV-1 Replication by a Combination of Endonucleolytic Ribozymes (RNase P and tRNase ZL)

We examined the combinatorial action of RNase P and tRNase ZL-mediated specific inhibition of HIV-1 in cultured cells. We designed two short extra guide sequences (sEGS) that specifically recognize the tat and vif regions of HIV-1 mRNA and mediate the subsequent cleavage of hybridized mRNA by the RNase P and tRNase ZL components. We constructed an RNase P and tRNase ZL-associated vif and tat sEGS expression vector, which used the RNA-polymerase III dependent U6 promoter, as an expression cassette for EGS. Together, the RNase P and tRNase ZL-associated sEGS molecules allow more efficient suppression of HIV-1 mRNA production when separately applied. The possibilities offered by the vector to encode sEGS will provide a powerful tool for gene therapy.     

Differential regulation of host cellular genes by HIV-1 viral protein R (Vpr): cDNA microarray analysis using isogenic virus

HIV-1 Vpr is a protein with multiple functions. It has been suggested that such pleiotropic effects by a viral protein may be mediated by its association with viral and cellular proteins or through modulation of expression of specific cellular genes. To address this, we used cDNA microarray techniques to analyze the regulation of a panel of host cellular genes by HIV-1 Vpr using isogenic HIV-1 either with or without Vpr expression. Results indicate that Vpr downregulated the expression of genes involved in cell cycle/proliferation regulation, DNA repair, tumor antigens, and immune activation factors, and upregulated many ribosomal and structural proteins. These results for the first time reveal the involvement of several potential cellular genes, which may be useful, both for understanding Vpr functions and for the development of therapeutics targeting the Vpr molecule.     

The Conformation and Orientation of a 27-Residue CCR5 Peptide in a Ternary Complex with HIV-1 gp120 and a CD4-Mimic Peptide

Interaction of CC chemokine receptor 5 (CCR5) with the human immunodeficiency virus type 1 (HIV-1) gp120/CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of two to four tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied both in its free form and in a ternary complex with deglycosylated gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27), which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by 2-fold the length of Nt-CCR5's known structure. Two-dimensional saturation transfer experiments and measurement of relaxation times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4, while the C-terminal segment of Nt-CCR5(1-27) points toward the target cell membrane, reflecting an Nt-CCR5 orientation that differs by 180° from that of a previous model. A gp120 site that could accommodate ^CCR5Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated ^CCR5Tyr14 with ^gp120Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion, furthering our understanding of CCR5 recognition by HIV-1.     

Differentiation of Coxiella burnetii by Sequence Analysis of the Gene (com1) Encoding a 27-kDa Outer Membrane Protein

The gene (com1) encoding a 27-kDa outer membrane protein in 21 strains of Coxiella burnetii from a variety of clinical and geographical sources was sequenced for strain differentiation. The com1 gene was highly conserved among all the strains tested but there were several differences in nucleotide and deduced amino acid sequences. Based on the com1 gene-specific nucleotides and deduced amino acids, the 21 strains were divided into four groups. Group 1 contained 14 strains originating from ticks, cattle and human cases of acute Q fever. Groups 2 and 3 included 2 and 3 strains, respectively, originating from human cases of chronic Q fever. Group 4 contained 2 strains originating from a human case of acute Q fever and a goat with abortion. The results indicated that the strains originating from ticks, cattle and human cases of acute Q fever differed at the molecular level from those of human chronic Q fever. This study suggests that a sequence analysis of the com1 gene can be used for strain differentiation of C. burnetii.     

Involvement of the second extracellular loop and transmembrane residues of CCR5 in inhibitor binding and HIV-1 fusion: insights into the mechanism of allosteric inhibition

C-C chemokine receptor 5 (CCR5), a member of G-protein-coupled receptors, serves as a coreceptor for human immunodeficiency virus type 1 (HIV-1). In the present study, we examined the interactions between CCR5 and novel CCR5 inhibitors containing the spirodiketopiperazine scaffolds AK530 and AK317, both of which were lodged in the hydrophobic cavity located between the upper transmembrane domain and the second extracellular loop (ECL2) of CCR5. Although substantial differences existed between the two inhibitors—AK530 had 10-fold-greater CCR5-binding affinity (K_d = 1.4 nM) than AK317 (16.7 nM)—their antiviral potencies were virtually identical (IC₅₀ = 2.1 nM and 1.5 nM, respectively). Molecular dynamics simulations for unbound CCR5 showed hydrogen bond interactions among transmembrane residues Y108, E283, and Y251, which were crucial for HIV-1-gp120/sCD4 complex binding and HIV-1 fusion. Indeed, AK530 and AK317, when bound to CCR5, disrupted these interhelix hydrogen bond interactions, a salient molecular mechanism enabling allosteric inhibition. Mutagenesis and structural analysis showed that ECL2 consists of a part of the hydrophobic cavity for both inhibitors, although AK317 is more tightly engaged with ECL2 than AK530, explaining their similar anti-HIV-1 potencies despite the difference in K_d values. We also found that amino acid residues in the β-hairpin structural motif of ECL2 are critical for HIV-1-elicited fusion and binding of the spirodiketopiperazine-based inhibitors to CCR5. The direct ECL2-engaging property of the inhibitors likely produces an ECL2 conformation, which HIV-1 gp120 cannot bind to, but also prohibits HIV-1 from utilizing the “inhibitor-bound” CCR5 for cellular entry—a mechanism of HIV-1's resistance to CCR5 inhibitors. The data should not only help delineate the dynamics of CCR5 following inhibitor binding but also aid in designing CCR5 inhibitors that are more potent against HIV-1 and prevent or delay the emergence of resistant HIV-1 variants.     

The mechanism of alkylation of DNA by 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC), a metabolite of DIC (NSC-45388). Non-involvement of diazomethane

Comparison of the nuclear magnetic resonance spectra of chemically synthesized methyl-d₁-methanol with the methanol produced in the solvolytic decompostion of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC) in D₂O under acidic, basic or neutral conditions indicated that no deuterium was exchanged for the hydrogens on the methyl group. Diazomethane can therefore be ruled out as an intermediate in this reaction.The methyl-d₃-guanine isolated after incubation of methyl-d₃-MIC with calfthymus DNA in vitro displayed, on chemical ionization mass spectrometry, a quasimolecular ion (MH⁺) at m/e 169, which was 3 mass units higher than the quasimolecular ion for an undeuterated 7-methylguanine standard. The major fragment ions for 7-methyl-d₃-guanine on electron impact mass spectrometry likewise were situated at positions 3 mass units higher than the fragment ions for 7-methylguanine itself.These data indicate that the methylation of biological macromolecules by MIC must involve the transfer of an intact methyl group.     

Analysis of a novel antiinflammatory agent, 1-(7-tert.-butyl-2,3-dihydro-3,3-dimethylbenzo[b]furan-5-yl)-4-cyclopropylbutan-1-one (PGV-20229), in plasma matrices by stable-isotope-dilution gas chromatography-mass spectrometry

A sensitive and selective GC-MS method was developed for the determination of low levels of a novel antiinflammatory agent, 1-(7-tert.-butyl-2,3-dihydro-3,3-dimethylbenzo[b]furan-5-yl)-4-cyclopropylbutan-1-one (I), in small volumes of animal plasma. The method involved the addition of ¹³C₆-labeled-I to plasma samples, followed by a simple liquid-liquid extraction with hexane to isolate the analytes from matrix components. The levels of I in the sample extracts were determined by isotope-dilution GC-MS analysis using selected-ion monitoring. The method was linear over three orders of magnitude, with a limit of quantitation of 1.8 ng/ml I, using plasma sample volumes of 0.1 ml. The method was utilized to determine the pharmacokinetic parameters of I in rats and dogs, following intravenous administration.     

The Human IGF1R IRES likely operates through a Shine–Dalgarno‐like interaction with the G961 loop (E‐site) of the 18S rRNA and is kinetically modulated by a naturally polymorphic polyU loop

IGF1R is a proto‐oncogene with potent mitogenic and antiapoptotic activities, and its expression must be tightly regulated to maintain normal cellular and tissue homeostasis. We previously demonstrated that translation of the human IGF1R mRNA is controlled by an internal ribosome entry site (IRES), and delimited the core functional IRES to a 90‐nucleotide segment of the 5′‐untranslated region positioned immediately upstream of the initiation codon. Here we have analyzed the sequence elements that contribute to the function of the core IRES. The Stem2/Loop2 sequence of the IRES exhibits near‐perfect Watson–Crick complementarity to the G961 loop (helix 23b) of the 18S rRNA, which is positioned within the E‐site on the platform of the 40S ribosomal subunit. Mutations that disrupt this complementarity have a negative impact on regulatory protein binding and dramatically decrease IRES activity, suggesting that the IGF1R IRES may recruit the 40S ribosome by a eukaryotic equivalent of the Shine–Dalgarno (mRNA–rRNA base‐pairing) interaction. The homopolymeric Loop3 sequence of the IRES modulates accessibility and limits the rate of translation initiation mediated through the IRES. Two functionally distinct allelic forms of the Loop3 poly(U)‐tract are prevalent in the human population, and it is conceivable that germ‐line or somatic variations in this sequence could predispose individuals to development of malignancy, or provide a selectable growth advantage for tumor cells. J. Cell. Biochem. 110: 531–544, 2010. © 2010 Wiley‐Liss, Inc.     

One‐pot synthesis of (R)‐1‐(pyridin‐4‐yl)ethyl acetate using tandem catalyst prepared by co‐immobilization of palladium and lipase on mesoporous foam: Optimization and kinetic modeling

The synthesis of (R )‐1‐(pyridin‐4‐yl)ethyl acetate was achieved over tandem palladium‐lipase catalyst with 100% selectivity using 4‐acetyl pyridine as a reactant. The 2% w /w palladium and lipase catalyst was successfully co‐immobilized in the microenvironment of the mesocellular foam and characterized by various techniques. The palladium metal from catalyst hydrogenated 4‐acetyl pyridine to form 1‐(pyridin‐4‐yl)ethanol. The generated intermediate product then underwent kinetic resolution over lipase and selectively gave (R )‐1‐(pyridin‐4‐ yl)ethyl acetate. The catalytic conditions were then studied for optimal performance of both steps. The reaction conditions were optimized to 50 °C and toluene as a solvent. Both chemical and enzymatic kinetic models of the reaction were developed for a given set of reaction conditions and kinetic parameters were predicted. At optimal conditions, the obtained selectivity of intermediate (1‐(pyridin‐4‐yl)ethanol) was 51.38%. The final product yield of ((R )‐1‐(pyridin‐4‐yl)ethyl acetate) was 48.62%.     

An idic(15) associated with POF (premature ovarian failure): molecular cytogenetic definition of a case and review of the literature

We report on a 36-year-old infertile woman, presenting a premature ovarian failure with an otherwise normal female phenotype. Cytogenetic analyses showed the presence of a supernumerary marker chromosome, that was characterized by FISH (fluorescent in situ hybridization) and array CGH (comparative genomic hybridization). This marker chromosome was derived from chromosome 15, and contained only heterochromatic material. The Prader Willi/Angelman region was not present. No duplications of the 15q regions were detected by array CGH. Supernumerary markers of chromosome 15 have been reported in cases of infertility and amenorrhea, that is also described in cases with marker derived by other acrocentric chromosomes. The case here presented constitutes a further example that etiology of POF is not always associated with a defective gene, but in some cases oocytes atresia can be the consequence of the abnormal meiotic pairing of chromosomes.     

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.