RESUMO
To evaluate the potential that multiply resistant human immunodeficiency virus type 1 variants may arise during combination nucleoside and nonnucleoside reverse transcriptase inhibitor therapy, we constructed a series of mutant reverse transcriptase enzymes and viruses that coexpressed various combinations of resistance-associated amino acid substitutions. Substitutions at residues 100 (Leu-->Ile) and 181 (Tyr-->Cys), which mediate resistance to the nonnucleosides, suppressed resistance to 3'-azido-3'-deoxythymidine (AZT) when coexpressed with AZT-specific substitutions. However, a number of viral variants that exhibited significantly reduced susceptibilities to both classes of inhibitors were constructed.
Assuntos
HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa , Resistência Microbiana a Medicamentos , Transcriptase Reversa do HIV , Mutação , Relação Estrutura-Atividade , Zidovudina/farmacologiaRESUMO
The human immunodeficiency virus type 1 (HIV-1) protease is a homodimeric aspartyl endopeptidase that is required for virus replication. A number of specific, active-site inhibitors for this enzyme have been described. Many of the inhibitors exhibit significant differences in activity against the HIV-1 and HIV type 2 (HIV-2) enzymes. An initial study was conducted to ascertain the HIV-1 protease's potential to lose sensitivity to several test inhibitors while retaining full enzymatic activity. The substrate binding sites of the HIV-1 and HIV-2 enzymes are almost fully conserved, except for four amino acid residues at positions 32, 47, 76, and 82. Accordingly, recombinant mutant type 1 proteases were constructed that contained the cognate type 2 residue at each of these four positions. The substitution at position 32 resulted in a significant adverse effect on inhibitor potency. However, this substitution also mediated a noted increase in the Km of the substrate. Individual substitutions at the remaining three positions, as well as a combination of all four substitutions, had very little effect on enzyme activity or inhibitor susceptibility. Hence, the four studied active site residues are insufficient to be responsible for differences in inhibitor sensitivity between the HIV-1 and HIV-2 proteases and are unlikely to contribute to the generation of inhibitor-resistant mutant HIV-1 protease.
Assuntos
Aminoácidos/genética , Inibidores da Protease de HIV/farmacologia , Protease de HIV/genética , Benzopiranos/farmacologia , Sítios de Ligação/genética , Clonagem Molecular , Gráficos por Computador , Resistência Microbiana a Medicamentos , Escherichia coli/genética , Protease de HIV/metabolismo , Inibidores da Protease de HIV/química , Isoquinolinas/farmacologia , Cinética , Modelos Moleculares , Morfolinas/farmacologia , Peptídeos/farmacologia , Quinolinas/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saquinavir , Valina/análogos & derivados , Valina/farmacologiaRESUMO
The nonnucleoside reverse transcriptase (RT) inhibitors comprise a class of structurally diverse compounds that are functionally related and specific for the human immunodeficiency virus type 1 RT. Viral variants resistant to these compounds arise readily in cell culture and in treated, infected human. Therefore, the eventual clinical usefulness of the nonnucleoside inhibitors will rely on a thorough understanding of the genetic and biochemical bases for resistance. A study was performed to assess the effects of substitutions at each RT amino acid residue that influences the enzyme's susceptibility to the various nonnucleoside compounds. Single substitutions were introduced into both purified enzyme and virus. The resulting patterns of resistance were markedly distinct for each of the tested inhibitors. For instance, a > 50-fold loss of enzyme susceptibility to BI-RG-587 was engendered by any of four individual substitutions, while the same level of relative resistance to the pyridinone derivatives was mediated only by substitution at residue 181. Similarly, substitution at residue 181. Similarly, substitution at residue 106 had a noted effect on virus resistance to BI-RG-587 but not to the pyridinones. The opposite effect was mediated by a substitution at residue 179. Such knowledge of nonucleoside inhibitor resistance profiles may help in understanding the basis for resistant virus selection during clinical studies of these compounds.
Assuntos
Antivirais/farmacologia , HIV-1/genética , Mutação/genética , Nucleosídeos/farmacologia , DNA Polimerase Dirigida por RNA/genética , Inibidores da Transcriptase Reversa , Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Antivirais/uso terapêutico , Benzoxazóis/uso terapêutico , DNA Viral/genética , Variação Genética , Transcriptase Reversa do HIV , HIV-1/enzimologia , Humanos , Piridonas/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Several novel, structurally distinct classes of specific human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) nonnucleoside inhibitors have been described recently. These include the pyridinone derivatives L-697,639, L-697,661, and L-696,229 as well as BI-RG-587 and the tetrahydroimidazo[4,5,1-j,k]-benzodiazepin-2(1H)-one and -thione compounds. Previous studies have implicated involvement of the RT amino acid residues at positions 103, 181, and 188 in the activity of the compounds. Accordingly, HIV-1 RT mutants containing a series of amino acid substitutions at these positions were constructed. The relative resistance of purified mutant enzymes to each of the inhibitors was assessed. This analysis established the functional equivalence of the three inhibitor classes and provided evidence for the interaction of the 103 site with the 181/188 region. Amino acid substitutions at these positions were also found to influence RT sensitivity to inhibition by phosphonoformate, thereby suggesting a close association between this pyrophosphate analog's binding site in RT and the binding site of the nonnucleoside inhibitors. In addition, aromatic stacking of the amino acid side groups at residues 181 and 188 was suggested to be required for inhibitor activity.
Assuntos
Antivirais/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Inibidores da Transcriptase Reversa , Sequência de Aminoácidos , Azepinas/farmacologia , Benzodiazepinas/farmacologia , Benzoxazóis/farmacologia , Resistência Microbiana a Medicamentos/genética , Transcriptase Reversa do HIV , Imidazóis/farmacologia , Cinética , Mutagênese Sítio-Dirigida , Nevirapina , Piridinas/farmacologia , Piridonas/farmacologia , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/isolamento & purificação , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is potently inhibited by a structurally diverse group of nonnucleoside compounds. These include pyridinone derivatives, tetrahydroimadazo[4,5,1-j,k][1,4]-benzodiazepin-2(1H)-one and -thione, and BI-RG-587 (nevirapine). The compounds act noncompetitively, by an unknown mechanism, with respect to template-primer and nucleotide substrates. Despite a high degree of similarity between the HIV-1 and HIV-2 RTs, the HIV-2 enzyme is totally insensitive to these inhibitors. Using a novel method for joining DNA sequences, we have exploited this difference between the two enzymes to identify the regions of the RT that contribute to the compounds' inhibitory activities. The relative in vitro sensitivities of HIV-1/HIV-2 chimeric and site-specific mutant enzymes were determined. Sensitivity to inhibition was largely, though not exclusively, dependent upon the RT region defined by amino acid residues 176 to 190, with specific contributions by residues 181 and 188. The region defined by residues 101 to 106 was found to functionally interact with the domain from 155 to 217. In addition, the functional equivalence of the three inhibitor groups was shown.
Assuntos
Antivirais/farmacologia , Azepinas/farmacologia , Benzodiazepinas/farmacologia , Benzoxazóis/farmacologia , HIV-1/enzimologia , Imidazóis/farmacologia , Piridinas/farmacologia , Piridonas/farmacologia , Inibidores da Transcriptase Reversa , Resistência Microbiana a Medicamentos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Transcriptase Reversa do HIV , Humanos , Nevirapina , FenótipoRESUMO
Bacterially expressed Tat protein of human immunodeficiency virus type 1 binds selectively to short RNA transcripts containing the viral transactivation-responsive element (TAR). Sequences sufficient for Tat interaction map to the distal portion of the TAR stem-loop. We show that critical sequences for Tat binding are located in the single-stranded "bulge," but no requirement for specific "loop" sequences could be demonstrated. TAR RNA competed for complex formation, and TAR mutants exhibited up to 10-fold reduced affinity for Tat. Synthetic peptides containing the basic region of Tat bound selectively to TAR RNA and exhibited the same sequence requirements and similar relative affinities for mutant TAR RNA as the intact protein. These results suggest that Tat contains a small RNA-binding domain capable of recognizing TAR and implicate functional relevance for direct Tat-TAR interaction in transactivation.
Assuntos
Produtos do Gene tat/metabolismo , Repetição Terminal Longa de HIV/genética , HIV-1/genética , Sequências Reguladoras de Ácido Nucleico , Sequência de Aminoácidos , Ligação Competitiva , Análise Mutacional de DNA , Técnicas In Vitro , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes , Relação Estrutura-Atividade , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Human rhinoviruses are the major causative agents of the common cold in humans and have been divided into major and minor groups based on receptor specificity. cDNAs encoding the light and heavy chains of a murine monoclonal antibody that recognizes the major group receptor were cloned, abundantly expressed in Escherichia coli, and renatured into Fab fragments that blocked virus binding and protected HeLa cell monolayers from rhinovirus infection. Elimination of the cysteines normally bridging the heavy and light chains yielded molecules indistinguishable from wild-type Fab fragments in virus binding assays. Single-chain antibodies with covalently linked light and heavy variable domains were also expressed and showed receptor binding and cell protection activities. These recombinant antibody fragments are potentially useful in preventing or treating common colds in humans.
Assuntos
Genes de Imunoglobulinas , Fragmentos Fab das Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Receptores Virais/fisiologia , Rhinovirus/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Transformação Celular Viral , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Células HeLa/fisiologia , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/isolamento & purificação , Dados de Sequência Molecular , Conformação Proteica , Receptores Virais/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Rhinovirus/genética , Rhinovirus/fisiologiaRESUMO
The relative contribution of thymus-derived lymphocytes (T-cells) and of macrophages to resistance to primary infection with herpes simplex virus type 1 (HSV-1) was studied in C58 mice. Resistance was dependent on macrophage competence, but was relatively independent of T-lymphocyte competence. Although aging mice became progressively more deficient in functional T-cells, as demonstrated by a decreasing resistance to transplanted line Ib leukemia and by declining responses to T-cell nitogens (concanavalin A and phytohemagglutinin), their resistance to HSV-1 increased with increasing age. Moreover, in mice that were made selectively deficient in T-cells by the combination of adult thymectomy and treatment with anti-thymocyte serum, resistance to HSV-1 did not correlate spleen and mesenteric lymph nodes. However, selective reduction of macrophages by intraperitoneal injection of silica resulted in enhanced susceptibility to HSV-1. Furthermore, in vitro suppression of HSV-1 plaque formation in mouse embryo fibroblast cells was obtained by cocultivation of infected fibroblast monolayers with peritoneal macrophages, but not with splenic lymphocytes, from adult mice. Macrophages from weanling mice failed to suppress the development of plaques, indicating that the increase in resistance to HSV-1 with age is a result of increased macrophage competence.