Dominique Coulaud

Efficient DNA Transfection Mediated by the C-Terminal Domain of Human Immunodeficiency Virus Type 1 Viral Protein R

ABSTRACT Viral protein R (Vpr) of human immunodeficiency virus type 1 is produced late in the virus life cycle and is assembled into the virion through binding to the Gag protein. It is known to play a significant role early in the viral life cycle by facilitating the nuclear import of the preintegration complex in nondividing cells. Vpr is also able to interact with nucleic acids, and we show here that it induces condensation of plasmid DNA. We have explored the possibility of using these properties in DNA transfection experiments. We report that the C-terminal half of the protein (Vpr52–96) mediates DNA transfection in a variety of human and nonhuman cell lines with efficiencies comparable to those of the best-known transfection agents. Compared with polylysine, a standard polycationic transfection reagent, Vpr52–96 was 10- to 1,000-fold more active. Vpr52–96-DNA complexes were able to reach the cell nucleus through a pH-independent mechanism. These observations possibly identify an alternate pathway for DNA transfection.

The Cationic Amphipathic α-Helix of HIV-1 Viral Protein R (Vpr) Binds to Nucleic Acids, Permeabilizes Membranes, and Efficiently Transfects Cells

Viral protein R (Vpr) is a small protein of 96 amino acids that is conserved among the lentiviruses human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. We recently sought to determine whether the karyophilic properties of Vpr, as well as its ability to bind nucleic acids, could be used to deliver DNA into cells. We have found that the C-terminal domain of Vpr-(52–96) is able to efficiently transfect various cell lines. Here, we show that the shortest active sequence for gene transfer corresponds to the domain that adopts a α-helix conformation. DNA binding studies and permeabilization assays performed on cells demonstrated that the peptides that are efficient in transfection condense plasmid DNA and are membranolytic. Electron microscopy studies and transfection experiments performed in the presence of inhibitors of the endocytic processes indicated that the major entry pathway of Vpr-DNA complexes is through endocytosis. Taken together, the results show that the cationic C-terminal α-helix of Vpr has DNA-condensing as well as membrane-destabilizing capabilities, both properties that are indispensable for efficient DNA transfection.

Histidine Containing Peptides and Polypeptides as Nucleic Acid Vectors

Nucleic acid transfer in mammalian cells is drastically improved with devices which increase their delivery in the cytosol upon endocytosis. In this chapter, we describe the effect on plasmid DNA (pDNA) and oligonucleotide (ODN) transfer, of an histidine-rich peptide (H5WYG), histidylated oligolysine (HoK), and histidylated polylysine (HpK) designed on the basis of the membrane destabilization capacity of poly-L-histidine at a pH close to that of the endosomes. We report that H5WYG, which permeabilizes the cell membrane at pH 6.4, favors the transfection mediated by lactosylated polylysine/pDNA complexes and, by lowering the pH of extracellular medium, allows the loading of the cytosol and the cell nucleus with ODN. We show that HoK forms small cationic spherical particles of 35 nm with ODN and HpK rod or toroid cationic particles of 100 nm with pDNA. PEGylation stabilizes these particles at physiological salt concentration. We also show that (i) HoK/ODN complexes yield a more than 20-fold increase of the biological activity of antisense ODN towards the inhibition of transient as well as constitutive gene expression and (ii) HpK/pDNA complexes yield a transfection efficiency of 3–4.5 order of magnitude higher than do polylysine/pDNA complexes. We also provide evidence that the effect of these polyhistidylated molecules is mediated by imidazole protonation in endosomes. Overall our data show that polyhistidylated molecules constitute interesting devices for an efficient cytosolic delivery of nucleic acids, and that ionic complexes between histidylated polylysine and a pDNA are attractive for developing a nonviral gene delivery system.

Human Immunodeficiency Virus Type 1 Central DNA Flap: Dynamic Terminal Product of Plus-Strand Displacement DNA Synthesis Catalyzed by Reverse Transcriptase Assisted by Nucleocapsid Protein

ABSTRACT To terminate the reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome, a final step occurs within the center of the proviral DNA generating a 99-nucleotide DNA flap (6). This step, catalyzed by reverse transcriptase (RT), is defined as a discrete strand displacement (SD) synthesis between the first nucleotide after the central priming (cPPT) site and the final position of the central termination sequence (CTS) site. Using recombinant HIV-1 RT and a circular single-stranded DNA template harboring the cPPT-CTS sequence, we have developed an SD synthesis-directed in vitro termination assay. Elongation, strand displacement, and complete central flap behavior were analyzed using electrophoresis and electron microscopy approaches. Optimal conditions to obtain complete central flap, which ended at the CTS site, have been defined in using nucleocapsid protein (NCp), the main accessory protein of the reverse transcription complex. A full-length HIV-1 central DNA flap was then carried out in vitro. Its synthesis appears faster in the presence of the HIV-1 NCp or the T4-encoded SSB protein (gp32). Finally, a high frequency of strand transfer was shown during the SD synthesis along the cPPT-CTS site with RT alone. This reveals a local and efficient 3′-5′ branch migration which emphasizes some important structural fluctuations within the flap. These fluctuations may be stabilized by the NCp chaperone activity. The biological implications of the RT-directed NCp-assisted flap synthesis are discussed within the context of reverse transcription complexes, assembly of the preintegration complexes, and nuclear import of the HIV-1 proviral DNA to the nucleus toward their chromatin targets.

Characterization of Phytomonas sp. kinetoplast DNA: A plant pathogenic trypanosomal species

Phytomonas sp. which belongs to the Trypanosomatidae family, is a pathogen of plants. Its kinetoplast DNA consists of a huge network of about 7000 catenated minicircles of 2880 ± 30 base pairs each. It represents 30–33% of the total cellular DNA. An AT composition of 65% has been evaluated from the buoyant density xxx = 1.694 . The restriction endonuclease HpaII was the only one able to cleave the minicircles into a single fragment. The other enzymes so far tested (EcoRI, HindIII, HaeIII, BamHI) do not cleave these minicircles significantly. Analysis of the kDNA by Southern blot hybridization shows that minicircles of Phytomonas sp. have little sequence homology with minicircles of other species of trypanosomes. Maxicircles, present in low proportions, have been identified. Our data indicate that the kinetoplast DNA of Phytomonas sp. presents biochemical characteristics which could be used to identify the Phytomonas genus.

Kinetoplast DNA permits characterization of pathogenic plant trypanosomes of economic importance

Summary— Twelve Phytomonas isolates were obtained from different plants originating from several countries and cultured in vitro in complex media. The kinetoplast DNA (kDNA) was purified and observed by electron microscopy. The structure of kDNA from all isolates appeared as a large network of interlocked minicircles with some maxicircles extruding from the network, as has often been shown for Trypanosomatidae. Topoisomerase II resolved the kDNA network into free minicircles which were then analyzed by electron microscopy and by electrophoresis in agarose gel. The minicircle sizes varied from 1.3 to 2.8 kilobase pairs according to the Phytomonas isolate. The analysis by restriction endonucleases revealed a base sequence heterogeneity in the minicircles of 10 of these Phytomonas isolates. By contrast, in 2 Phytomonas isolates, more than 90% of their minicircle content was found to be homogeneous. Most interestingly, the minicircle cleavage patterns were found to be different between Phytomonas isolates and thus could be used to distinguish them.

Selective alteration of mitochondrial function by ditercalinium (NSC 335153), a DNA bisintercalating agent

The bifunctional intercalator Ditercalinium (NSC 335153) demonstrates an anti-tumoral cytotoxicity markedly different from other intercalating agents. A delayed toxicity is observed in eucaryotic cells, both in vitro and in vivo, at drug concentrations far below those required to observe immediate toxic effects. Fluorescence microscopy demonstrates that Ditercalinium and the mitochondrial-staining fluorophore DiOC2(5) are concentrated in the same cellular organelles of L1210 cells. Electron microscopy of Ditercalinium-treated cells reveals extensive and progressive swelling of mitochondria, with no other ultrastructural changes observed. Ditercalinium uptake and toxicity are in part related to mitochondrial membrane potential. However, drug accumulation itself does not immediately alter the mitochondrial membrane potential. Cellular ATP pool levels and the rate of respiration fall progressively after drug treatment. Nucleotide pools in DC3F cells, measured between drug treatment and death, show marked drops in pyrimidine levels while purine nucleotide levels decline more slowly. Addition of uridine or cytidine partially rescues Ditercalinium-treated cells, while toxicity is increased in the presence of 2-deoxyglucose. The combined evidence indicates that the toxicity of Ditercalinium to murine leukemia cells (L1210) and Chinese Hamster lung cells (DC3F) is due to disruption of mitochondrial function.

Electron Microscopic Observations of the Effects of Anthraquinone Derivatives on Plasmid DNA

Electron microscopy was used to analyse the precipitation of DNA observed when mixed with two tripeptide derivatives of mitoxantrone, with or without a 5,8-dihydroxy group (DHQ-GHK and Q-GHK, respectively) on the anthraquinonic ring. This precipitation was compared to that obtained with the basic drugs, mitoxantrone (DHAQ) and ametantrone (AQ). The effects of these compounds on the supercoiling of form I and the lengthening of form II of pBR322 DNA molecules, respectively, were evaluated. A strong lengthening of the DNA molecules was observed for ametantrone (max: 57%), but only 32% for Q-GHK, both at r (drug/base pari) = 250. With the dihydroxy derivative DHQ-GHK, it was not possible to show more than a 10% increase in length because DNA molecules were not measurable at r greater than 100. Only Q-GHK relaxed supercoiled molecules at the low r values of 10. Complex phenomena of condensation-precipitation were observed with these two tripeptide derivatives. In addition to a strong lengthening of form II DNA molecules, AQ induced specifically the formation of toruses, and DHAQ that of large organized DNA condensation. The variety of the aggregations is described and discussed with regard to the antitumor properties of these derivatives, and the literature concerning the various descriptions of DNA aggregation.

Electron microscopy of macromolecules. The French contribution.

Since 1966, French laboratories have contributed original data on the electron microscope (EM) observation of isolated biological macromolecules, as well as on the conditions needed for the optimization of such observations. We shall present briefly the different progresses in the preparation techniques in combination with a rapid description of the main results. A short review of recent results will show the latest orientations of the French EM research at the molecular level.