In VitroSelection for Different Mutational Patterns in the HIV-1 Reverse Transcriptase Using High and Low Selective Pressure of the Nonnucleoside Reverse Transcriptase Inhibitor HBY 097

AbstractIn vitroresistance of HIV-1 against high levels of HBY 097 ((S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4-dihydro-quinoxaline-2(1H)-thione) and other quinoxaline nonnucleoside reverse transcriptase inhibitors (NNRTIs) is characterized by a specific amino acid substitution in the reverse transcriptase (RT), Gly190Glu. This change results in decreased RT polymerase activity and in reduced growth properties of the corresponding viral variant. Here we show that the appearance of the crippling mutation at codon 190 can be prevented by lowering the selective pressure exerted by HBY 097. Under low selective pressure an accumulation of other NNRTI-specific mutations is observed. Up to five NNRTI-specific substitutions were detected in some of these virus lineages. In addition, we report novel RT amino acid changes which were not observed previously, including Val106Ile, Val106Leu, and Gly190Thr. HBY 097 selects for different mutational patterns under high and low selective pressure conditions, respectively. Thus, the type of mutations which appear in HIV-infected patients undergoing therapy may be determined by the levels of the selecting drug

Die Biologie der Onkogene

Onkogene wurden zuerst als dominante, maligne transformierende Gene in den Genomen von Retroviren identifiziert. In normalen Zellen existieren eng verwandte Gene, die phylogenetisch hochkonserviert sind und wichtige regulatorische Funktionen bei Differenzierung und Wachstum haben. Diese Gene werden als Proto-Onkogene bezeichnet. Onkogene haben häufig ähnliche, Jedoch aberrante enzymatische oder regulatorische Aktivitäten wie die normalen Gene. Sie werden in verschiedene Klassen eingeteilt (Proteinkinasen, GTP-bindende Proteine, Wachstumsfaktor-Analoga, nukleare Proteine, Wachstumsfaktor-Rezeptor-Analoga). Die normalen Funktionen der Proto-Onkogene werden schon ansatzweise verstanden. So regeln beispielsweise die Analoga der Tyrosinkinase-Onkogene unter anderem die Hämatopoese. Ein neuer Vertreter dieser Gruppe wurde von uns isoliert und als c-tkl bezeichnet. Onkogene treten in malignen Geweben auch unabhängig von Retroviren auf. Mit DNA aus Tumoren gelang es in Transfektionsexperimenten, normale Zellen zu transformieren. Die Analyse zeigte, daß ein Onkogen in die Zellen eingeschleust worden war, das man schon von Experimenten mit Retroviren kannte. Gene, die das Transformations-Potential der Onkogene inhibieren, werden als Tumor-Suppressor-Gene bezeichnet und stellen einen neuen Zweig der Krebsforschung dar. Insgesamt ist die Entstehung einer Krebszelle immer an eine oder mehrere genetische Veränderungen gebunden, die ein regulatorisch wichtiges normales Gen funktionell ändern oder es zur falschen Zeit, in der falschen Zelle oder in der falschen Menge exprimieren. Diese genetische Änderung kann angeboren sein, sie kann durch die Infektion mit Viren, durch Strahlen, durch chemische Karzinogenese oder durch spontane Mutation erfolgen. Die Tatsache, daß heute eine Vielzahl von Genen bekannt ist, deren Veränderung Krebs induziert, läßt für die Zukunft eine wesentlich verbesserte Tumor-Diagnostik und eine spezifischere Therapie erwarten. Die systematische Aufklärung der Genome höherer Organismen dürfte für das Studium der Tumor-Suppressorgene und der Onkogene neue Erkenntnisse liefern.Oncogenes were originally discovered as single, dominantly transforming genes in the genomes of acutely transforming retroviruses. Proto-oncogenes are genes within normal cells, which are closely related to oncogenes. They exhibit normal metabolic functions in cells, but when altered genetically can act äs transforming genes (oncogenes). According to their biochemical functions, oncogenes are subdivided into different classes (protein kinases, GTP-binding proteins, growth-factor-analoga, nuclear proteins and analpga of hormone receptors). Recently, we isolated a new member of the tyrosine kinase tamily. Among other functions, this group of enzymes plays a key-role in the regulation of hematopoesis. The new gene was named c-tkl. Oncogenes are present in cancer cells also without infection by a retrovirus. While retroviral infection will introduce an aberrant gene, carcinogenesis without retroviruses occurs through alterations of the proto-oncogenes present within the cell. During the past years, anti-oncogenes were discovered. These recessive genes inhibit the transforming potential of oncogenes and failure of their function results in a cancer cell. Carcinogenesis is thus viewed äs the expression of either functionally altered genes, or the expression of a protooncogene at the wrong time, within the wrong cell or at a wrong ämount The genomic alterations leading to such changes can be inherited, introduced by viral infection; induced by chemical carcinogenesis, spontaneous mutation or radiation. It can be expected that the study of the genomes of higher organisms will shed further light on the function of tumor-suppressor genes äs well äs on proto-oncogenes and that knowledge of the genes leading to a cancer cell will greatly improve cancer diagnostic and eventually also therapy

Nef Proteins of Distinct HIV-1 or -2 Isolates Differ in Their Binding Properties for HCK: Isolation of a Novel Nef Binding Factor with Characteristics of an Adaptor Protein

AbstractThe Nef gene of the human and simian immunodeficiency viruses HIV and SIV has been implicated in pathogenicity; however, the mechanism by which Nef induces disease is still unknown. An impact on signal transduction in cells has been suggested by the interaction of Nef from an HIV-1 strain and tyrosine kinases like HCK and LCK as well as serine/threonine kinases. We have confirmed the binding of HCK to HIV-1 subtype B Nef and demonstrated an equally strong interaction with a subtype E Nef protein but weaker binding to Nef of HIV-2 subtype A (HIV-2D194). No binding, however, was observed to HIV-2 subtype B Nef (HIV-2D205). Instead, this protein bound to a novel cellular protein,Nefin1,with characteristics of an adaptor protein and strong expression in all human hematopoietic tissues.Nefin1binds through an amino-terminal domain, which is related to SH3 domains. For interaction of Nef withNefin1,the PxxP motif and the three-dimensional conformation of the molecule appear necessary. In conclusion, this study demonstrates that Nef proteins of divergent strains of HIV-1 and HIV-2 may use different elements of signal transduction pathways for the induction of pathogenicityin vivo

Virology, part 4 : specialty session transcript

Meeting: International Conference on AIDS, 5th, 4-9 June, 1989, Montreal, QC, CAPresenters: Françoise Bachelerie, Helga Rübsamen-Waigmann, James A. Hoxie, Paul J. Durda, Françoise Rey, Phalguni Gupt

Virology, part 4 : specialty session transcript

Meeting: International Conference on AIDS, 5th, 4-9 June, 1989, Montreal, QC, CAPresenters: Françoise Bachelerie, Helga Rübsamen-Waigmann, James A. Hoxie, Paul J. Durda, Françoise Rey, Phalguni Gupt

Novel Whole-Cell Antibiotic Biosensors for Compound Discovery▿

Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of ≤25 μg/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis

Biological Characterization of Novel Inhibitors of the Gram-Positive DNA Polymerase IIIC Enzyme

Novel N-3-alkylated 6-anilinouracils have been identified as potent and selective inhibitors of bacterial DNA polymerase IIIC, the enzyme essential for the replication of chromosomal DNA in gram-positive bacteria. A nonradioactive assay measuring the enzymatic activity of the DNA polymerase IIIC in gram-positive bacteria has been assembled. The 6-anilinouracils described inhibited the polymerase IIIC enzyme at concentrations in the nanomolar range in this assay and displayed good in vitro activity (according to their MICs) against staphylococci, streptococci, and enterococci. The MICs of the most potent derivatives were about 4 μg/ml for this panel of bacteria. The 50% effective dose of the best compound (6-[(3-ethyl-4-methylphenyl)amino]-3-{[1-(isoxazol-5-ylcarbonyl)piperidin-4-yl]methyl}uracil) was 10 mg/kg of body weight after intravenous application in a staphylococcal sepsis model in mice, from which in vivo pharmacokinetic data were also acquired