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1.
AIDS Res Hum Retroviruses ; 20(2): 135-44, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15018700

RESUMO

The retroviral integrase protein (IN) is essential for virus replication and, therefore, an attractive target for the development of inhibitors to treat human immunodeficiency virus (HIV) infection. Diverse classes of compounds that are active against this protein have been discovered using in vitro assays. Here we describe the synthesis of a novel compound, 3,8-dibromo-7-amino-4-hydroxy-2-naphthalenesulfonic acid (2BrNSA), which inhibits the in vitro activities of the full-length HIV-1 and avian sarcoma virus (ASV) integrases, and the isolated catalytic core fragment of the ASV protein (residues 52-207). The compound also inhibits retroviral reverse transcriptase in vitro, but the IC(50) for the HIV-1 enzyme is almost two orders of magnitude higher than for HIV-1 integrase. The inhibitor was found to be active in cell culture, preventing reporter gene transduction of HeLa cells by both ASV and HIV-1 vectors. Neither viral attachment nor uptake into cells appeared to be affected in these transfections, whereas accumulation of vector DNA and its joining to host DNA were both drastically reduced in the presence of the inhibitor. Propagation of two different strains of replication-competent HIV-1 in human peripheral blood mononuclear cells (PBMCs) was also reduced by the inhibitor, allowing survival of a substantial number of cells in the treated cultures. Based on these and other results we speculate that binding of 2BrNSA to integrase in infected cells interferes not only with its catalytic activity but also with critical interactions that are required for the formation or function of the reverse transcriptase complex. Its activity in cell culture suggests that this inhibitor may provide a valuable new lead for further development of drugs that target early steps in the HIV life cycle.


Assuntos
Inibidores de Integrase de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Naftalenos/farmacologia , Vírus do Sarcoma Aviário/efeitos dos fármacos , Vírus do Sarcoma Aviário/enzimologia , Vírus do Sarcoma Aviário/genética , Sequência de Bases , DNA Viral/genética , Inibidores de Integrase de HIV/química , HIV-1/genética , Células HeLa , Humanos , Técnicas In Vitro , Inibidores de Integrase/química , Inibidores de Integrase/farmacologia , Naftalenos/química , Transdução Genética , Replicação Viral/efeitos dos fármacos
2.
J Virol ; 75(19): 9549-52, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11533219

RESUMO

Formation of stably integrated proviruses is inefficient in cells that are defective in the cellular nonhomologous end-joining (NHEJ) DNA repair pathway (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644-647, 1999; R. Daniel, R. A. Katz, and A. M. Skalka, Mol. Cell. Biol. 21:1164-1172, 2001). However, the requirement for NHEJ function is not absolute, as 10 to 20% of infected NHEJ-deficient cells can express retrovirus- transduced reporter genes in a stable fashion. To learn more about the compensatory mechanism by which viral DNA may be incorporated into the host cell genome, we analyzed the nucleotide sequences of provirus-host DNA junctions in singly infected NHEJ-deficient cell clones. The results showed that the proviral DNA ends in all NHEJ-deficient clones had the normal 5'TG...CA3' sequence. In addition, 14 of the 19 proviruses analyzed were flanked by a 6-bp direct repeat of host sequences, as is characteristic for avian sarcoma virus integration. These results indicate that the DNA repair pathway which compensates for loss of NHEJ in these transductants does not introduce any gross abnormalities at the provirus-host DNA junctions.


Assuntos
DNA Viral/genética , DNA/genética , Infecções por Retroviridae/genética , Retroviridae/genética , Animais , Reparo do DNA , Humanos , Infecções por Retroviridae/virologia
3.
Mol Gen Mikrobiol Virusol ; (2): 30-3, 2000.
Artigo em Russo | MEDLINE | ID: mdl-10876765

RESUMO

Nucleotide sequence of Acholeplasma laidlawii genome site PG-8B (1000 n.p.), containing topoisomerase IV subunit genes (parE and parC), has been determined. Sequenced genome site contains a gene fragment coding for the C-terminal region of ParE and gene fragment coding for N-terminal region of ParC. Topoisomerase IV subunite genes in A. laidlawii genome are situated near each other and overlapping by 4 nucleotides. Selection in liquid nutrient medium with ascending antibiotic concentrations resulted in derivation of A. laidlawii PG-8B cells resistant to ciprofloxacin, a fluoroquinolone. The resistant clones contain a mutation in the parC QRDR region determining fluoroquinolone resistance: Ser(91) (corresponding to Ser(80) in Escherichia coli ParC) replacement) for Leu.


Assuntos
Acholeplasma laidlawii/enzimologia , Anti-Infecciosos/farmacologia , DNA Topoisomerases Tipo II/genética , Mutação , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , DNA Topoisomerase IV , DNA Topoisomerases Tipo II/química , Resistência Microbiana a Medicamentos/genética , Fluoroquinolonas , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
5.
Mol Gen Mikrobiol Virusol ; (4): 37-40, 1998.
Artigo em Russo | MEDLINE | ID: mdl-9987752

RESUMO

Single-strand conformation polymorphism (SSCP) method was used for nucleotide sequence variation analysis of the gyrase A subunit quinolone resistance determining region (gyrA QRDR) in a laboratory and clinical strains of M. hominis. The couple of primers selected for this region amplified specific product in clinical material. M. hominis cultures growing in the presence of different concentrations of ciprofloxacin were studied by the SSCP method. Ser(83) to Leu mutation described previously was detected in the presence of quinolone in concentrations of at least 10 mcg/ml. In addition, 11 clinical samples were tested. In all cases the results of SSCP were confirmed by direct sequencing of the region. In 2 cases the sequences of gyrA QRDR in clinical strains were the same as in the laboratory strain. A Ser(83)-Leu mutation was identified in 1 clinical sample, while in others nucleotide substitutes did not lead to changes in amino acid sequences. These data demonstrate high informative value of the SSCP method for evaluating nucleotide variation in gyrA QRDR and quinolone resistance of M. hominis.


Assuntos
Anti-Infecciosos/farmacologia , Mycoplasma hominis/efeitos dos fármacos , Sequência de Aminoácidos , Sequência de Bases , Ciprofloxacina/farmacologia , DNA Bacteriano , Resistência Microbiana a Medicamentos/genética , Dados de Sequência Molecular , Mutação , Mycoplasma hominis/genética , Polimorfismo Conformacional de Fita Simples , Homologia de Sequência de Aminoácidos
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