ABSTRACT
The DNA polymerase of the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a target widely used to inhibit HIV-1 replication. In contrast, very few inhibitors of the RNase H activity associated with RT have been described, despite the crucial role played by this activity in viral proliferation. DNA ligands with a high affinity for the RNase H domain of HIV-1 RT were isolated by systematic evolution of ligands by an exponential enrichment strategy (SELEX), using recombinant RTs with or without the RNase H domain. The selected oligonucleotides (ODNs) were able to inhibit in vitro the HIV-1 RNase H activity, while no effect was observed on cellular RNase H. We focused our interest on two G-rich inhibitory oligonucleotides. Model studies of the secondary structure of these ODNs strongly suggested that they were able to form G-quartets. In addition to the inhibition of HIV-1 RNase H observed in a cell free system, these ODNs were able to strongly diminish the infectivity of HIV-1 in human infected cells. Oligonucleotides described here may serve as leading compounds for the development of specific inhibitors of this key retroviral enzyme activity.
Subject(s)
Anti-HIV Agents/pharmacology , DNA, Viral/chemistry , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , Oligodeoxyribonucleotides/pharmacology , Ribonuclease H/antagonists & inhibitors , Virus Replication/drug effects , Base Sequence , CD4 Antigens/physiology , Cell Line , Drug Design , Gene Library , HIV Reverse Transcriptase/antagonists & inhibitors , HIV Reverse Transcriptase/chemistry , HIV-1/drug effects , HIV-1/physiology , Humans , Kinetics , Ligands , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Polymerase Chain Reaction , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ribonuclease H/chemistry , Transfection , beta-Galactosidase/geneticsABSTRACT
RNase H activities from HeLa cells (either of cytoplasmic or mitochondrial origin), and from mitochondria of beef heart and Xenopus ovaries, have been tested with RNA-DNA substrates of defined length (20 bp) and sequence. Substrates were either blunt-ended, or presented DNA or RNA overhangs. The hydrolysis profiles obtained at early times of the digestion showed a good correlation between the class of RNase H, either type I or II assigned according to biochemical parameters, whatever the organism. Consequently, the pattern of primary cuts can be considered as a signature of the predominant RNase H activity. For a given sequence, hydrolysis profiles obtained are similar, if not identical, for either blunt-ended substrates or those presenting overhangs. However, profiles showed variations depending on the sequence used. Of the three sequences tested, one appears very discriminatory, class I RNases H generating a unique primary cut 3 nt from the 3' end of the RNA strand, whereas class II RNases H generated two simultaneous primary cuts at 6 and at 8 nt from the 5' end of the RNA strand. Hydrolysis profiles further confirm the assignation of the mitochondrial RNase H activity from HeLa cells, beef heart and Xenopus oocytes to the class II.
Subject(s)
DNA/metabolism , Mitochondria/enzymology , RNA/metabolism , Ribonuclease H/metabolism , Animals , Cattle , Female , HeLa Cells , Humans , Hydrolysis , Mitochondria, Heart/enzymology , Nucleic Acid Hybridization , Ovary/enzymology , Xenopus laevisABSTRACT
The soilborne, vascular pathogen Ralstonia solanacearum, the causative agent of bacterial wilt, was shown to infect a range of Arabidopsis thaliana accessions. The pathogen was capable of infecting the Col-5 accession in an hrp-dependent manner, following root inoculation. Elevated bacterial population levels were found in leaves of Col-5, 4 to 5 days after root inoculation by the GMI1000 strain. Bacteria were found predominantly in the xylem vessels and spread systematically throughout the plant. The Nd-1 accession of A. thaliana was resistant to the GMI1000 strain of R. solanacearum. Bacterial concentrations detected in leaves of Nd-1, inoculated with an hrp+ strain of R. solanacearum, were only slightly higher than those detected in the susceptible accession, Col-5, following inoculation with a strain whose hrp gene cluster was deleted. Leaf inoculation of the GMI1000 strain on the resistant accession Nd-1 induced the formation of lesions in the older leaves of the rosette whereas the same strain of R. solanacearum provoked complete wilting of Col-5. Resistance to strain GMI1000 of R. solanacearum segregated as a simply inherited recessive trait in a genetic cross between Col-5 and Nd-1. F9 recombinant inbred lines generated between these two accessions were used to map a locus, RRS1, that was the major determinant of resistance between restriction fragment length polymorphism markers mi83 and mi61 on chromosome V. This region of the A. thaliana genome is known to contain many other pathogen recognition capabilities.
