RESUMEN
BMS-232632 is an azapeptide human immunodeficiency virus type 1 (HIV-1) protease (Prt) inhibitor that exhibits potent anti-HIV activity with a 50% effective concentration (EC(50)) of 2.6 to 5.3 nM and an EC(90) of 9 to 15 nM in cell culture. Proof-of-principle studies indicate that BMS-232632 blocks the cleavage of viral precursor proteins in HIV-infected cells, proving that it functions as an HIV Prt inhibitor. Comparative studies showed that BMS-232632 is generally more potent than the five currently approved HIV-1 Prt inhibitors. Furthermore, BMS-232632 is highly selective for HIV-1 Prt and exhibits cytotoxicity only at concentrations 6,500- to 23, 000-fold higher than that required for anti-HIV activity. To assess the potential of this inhibitor when used in combination with other antiretrovirals, BMS-232632 was evaluated for anti-HIV activity in two-drug combination studies. Combinations of BMS-232632 with either stavudine, didanosine, lamivudine, zidovudine, nelfinavir, indinavir, ritonavir, saquinavir, or amprenavir in HIV-infected peripheral blood mononuclear cells yielded additive to moderately synergistic antiviral effects. Importantly, combinations of drug pairs did not result in antagonistic anti-HIV activity or enhanced cytotoxic effects at the highest concentrations used for antiviral evaluation. Our results suggest that BMS-232632 may be an effective HIV-1 inhibitor that may be utilized in a variety of different drug combinations.
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacología , VIH-1/efectos de los fármacos , Oligopéptidos/farmacología , Piridinas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Sulfato de Atazanavir , Proteínas Sanguíneas , Células Cultivadas , Combinación de Medicamentos , Interacciones Farmacológicas , Productos del Gen gag/metabolismo , Humanos , Técnicas In Vitro , Pruebas de Sensibilidad Microbiana , Precursores de Proteínas/metabolismoRESUMEN
Using an assay capable of detecting sequence-specific RNA/protein interactions in mammalian cells, we demonstrate that the poliovirus and rhinovirus 3C proteinases are able to bind structured target RNA sequences derived from their respective 5' noncoding regions in vivo. Specific RNA binding by poliovirus 3C was found to be dependent on the integrity of stem-loop d of the RNA cloverleaf structure located at the 5' end of poliovirus genomic RNA. In contrast, mutation of stem-loop b did not prevent this in vivo interaction. However, mutation of stem-loop b, which serves as the RNA binding site for a cellular co-factor important for efficient poliovirus replication, did significantly attenuate the efficiency of 3C RNA binding in vivo and 3CD RNA binding in vitro. This in vivo protein:RNA binding assay was also used to identify several residues in 3C that are critical for RNA binding, but dispensable for 3C proteinase activity. The mammalian cell-based RNA binding assay described in this study may have considerable potential utility in the future detection or analysis of in vivo RNA/protein interactions unrelated to the 3C/RNA interaction described here.
Asunto(s)
Endopeptidasas/metabolismo , Picornaviridae/enzimología , ARN Viral/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Cloranfenicol O-Acetiltransferasa/genética , Endopeptidasas/genética , Productos del Gen tat/metabolismo , Genes Reporteros , Duplicado del Terminal Largo de VIH , Células HeLa , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Picornaviridae/genética , Poliovirus/enzimología , Poliovirus/genética , ARN Viral/química , ARN Viral/genética , Rhinovirus/enzimología , Rhinovirus/genética , Especificidad por Sustrato , Activación Transcripcional , TransfecciónRESUMEN
The transcription of genes carried by primate foamy viruses is dependent on two distinct promoter elements. These are the long terminal repeat (LTR) promoter, which regulates expression of the viral structural proteins, and a second internal promoter, located towards the 3' end of the env gene, that directs expression of the viral auxiliary proteins. One of these auxiliary proteins is a potent transcriptional transactivator, termed Bel-1 in human foamy virus (HFV) and Tas or Taf in the related simian foamy viruses, that is critical for foamy virus replication. Previously, it has been demonstrated that the LTR promoter element of HFV contains a DNA binding site for Bel-1 that is critical for transcriptional activation (F. He, W. S. Blair, J. Fukushima, and B. R. Cullen, J. Virol. 70:3902-3908, 1996). Here, we extended this earlier work by using methylation interference analysis to identify and characterize the Bel-1 DNA binding sites located in the HFV LTR and internal promoter elements. Based on these data, we propose a minimal, 25-bp DNA binding site for Bel-1, derived from the HFV internal promoter element, and show that this short DNA sequence mediates efficient Bel-1 binding both in vitro and in vivo. We further demonstrate that, as determined by both in vitro and in vivo assays, the Bel-1 target site located within the HFV internal promoter binds Bel-1 with a significantly higher affinity than the cap-proximal Bel-1 target site located in the LTR promoter. This result may provide a mechanistic explanation for the observation that the internal promoter is activated significantly earlier than the LTR promoter during the foamy virus life cycle.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de los Retroviridae/genética , Proteínas de los Retroviridae/metabolismo , Spumavirus/genética , Spumavirus/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Secuencia de Bases , Sitios de Unión/genética , ADN Viral/genética , ADN Viral/metabolismo , Genes Virales , Humanos , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Homología de Secuencia de Ácido Nucleico , Spumavirus/crecimiento & desarrolloRESUMEN
We describe a unique gain-of-function mutant of the TATA-binding protein (TBP) subunit of Saccharomyces cerevisiae TFIID that, at least in part, renders transcriptional transactivators dispensable for efficient mRNA expression. The yTBPN69S mutant enhances transcription from weaker yeast promoter elements by up to 50-fold yet does not significantly increase gene expression directed by highly active promoters. Therefore, this TBP mutant and transcriptional transactivators appear to affect a common rate-limiting step in transcription initiation. Consistent with the hypothesis that this step is TFIID recruitment, tethering of TBP to a target promoter via a heterologous DNA binding domain, which is known to bypass the need for transcriptional transactivators, also nullifies the enhancing effect exerted by the N69S mutation. These data provide genetic support for the hypothesis that TFIID recruitment represents a rate-limiting step in the initiation of mRNA transcription that is specifically enhanced by transcriptional transactivators.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , TATA Box , Factores de Transcripción TFII/genética , Factores de Transcripción/genética , Sitios de Unión , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Mutagénesis , ARN Mensajero/metabolismo , Proteína de Unión a TATA-Box , Transactivadores/metabolismo , Factor de Transcripción TFIID , Factores de Transcripción/metabolismo , Factores de Transcripción TFII/metabolismo , Transcripción GenéticaRESUMEN
The Bel-1 transcriptional transactivator encoded by human foamy virus (HFV) can efficiently activate gene expression directed by both the HFV long terminal repeat (LTR) and internal (Int) promoter elements. By DNA footprinting and gel retardation analysis, we demonstrate that Bel-1 can specifically bind to discrete sites in both the LTR and Int promoter elements in vitro. However, transactivation of the HFV LTR by Bel-1 was observed to require not only the promoter-proximal Bel-1 binding site identified in vitro but also additional promoter-distal sequences. These data suggest that Bel-1 binding is necessary but not sufficient for efficient transactivation of Bel-1-responsive promoters in mammalian cells and therefore raise the possibility that Bel-1 function may require the action of a cellular DNA binding protein(s). Importantly, these data demonstrate that Bel-1 is unique among retroviral regulatory proteins in being a sequence-specific DNA binding protein.
Asunto(s)
ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de los Retroviridae/metabolismo , Spumavirus/genética , Transactivadores/metabolismo , Secuencia de Bases , Células Cultivadas , ADN Viral/química , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Sequence and structure comparisons with homologous trypsin-like serine proteases have predicted the S1-specificity pocket in picornavirus 3C proteinases. In this study, we examine the putative roles of such residues in poliovirus 3C substrate recognition. Single amino acid substitutions at 3C residues Thr-142, His-161, Gly-163, Gly-164, and Ala-172 were introduced into near full-length poliovirus cDNAs, and protein processing was examined in the context of authentic 3C cis cleavage activity. Our data are consistent with residues Thr-142, His-161, Gly-163, and Gly-164 acting as important determinants of 3C substrate specificity and support published models of 3C protein structure. An in vivo analysis of mutant viruses containing individual amino acid substitutions at 3C residues Thr-142 and Ala-172 suggests that such residues are important determinants for viral RNA replication. In addition, bacterially expressed, recombinant 3CD polypeptides containing amino acid substitutions at Thr-142 and Ala-172 show altered RNA binding properties in mobility shift assays that use a synthetic RNA corresponding to the poliovirus 5'-terminal sequences.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Poliovirus/enzimología , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Virales , Proteasas Virales 3C , Sitios de Unión , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Escherichia coli , Células HeLa , Humanos , Metionina , Mutagénesis Sitio-Dirigida , Péptidos/metabolismo , Poliovirus/genética , Unión Proteica , Conformación Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por SustratoRESUMEN
The effects of activation domain synergy on transcription initiation and elongation have been examined utilizing a system that permits the targeting of a defined number of activation modules to promoter DNA. As predicted, incremental increases in targeted activation potential were found to result in corresponding increases in transcription initiation. Surprisingly, however, transcriptional processivity, and hence mRNA synthesis, required a threshold level of activation domain synergy that exceeded the level required for at least modest levels of transcription initiation. The degree to which transcriptional processivity was enhanced was shown to depend on the quantity of activation modules targeted to the promoter DNA, rather than the quality. While the RNA-sequence specific HIV-1 Tat trans-activator was also shown to enhance processivity in this assay system, Tat differed from DNA-sequence specific activation domains in exerting a more dramatic effect on the efficiency of transcript elongation.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Factores de Transcripción , Activación Transcripcional , Animales , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , ADN/genética , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Marcación de Gen , Genes Reporteros , Genes tat , VIH-1/genética , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/genética , TransfecciónRESUMEN
The potent C-terminal activation domain of the RelA (p65) subunit of the cellular transcription factor NF-kappa B is shown to contain several discrete acidic activation modules. These short, approximately 11-amino-acid modules were able to give rise to only a low level of transcription activation when fused to the GAL4 DNA-binding domain as monomers. However, dimers and higher-order multimers activated the transcription of minimal promoter elements as effectively as the full-length RelA or VP16 activation domain. Therefore, this 11-amino-acid RelA-derived acidic module appears to contain all of the sequence information required to fully activate a target promoter element as long as it is presented in a form that permits functional synergy. Critical primary sequence requirements for acidic activation module function included a core phenylalanine residue and flanking bulky hydrophobic residues. Overall negative charge was necessary but not sufficient for function. While dimeric forms of the 11-amino-acid acidic activation module bound to either TFIIB or TATA-binding protein efficiently in vitro, a similarly charged peptide lacking the core phenylalanine residue failed to interact. Overall, these data demonstrate that the biological activity of the RelA activation domain is dependent on acidic activator sequences that are closely comparable to those detected in the activation domain of the viral VP16 regulatory protein. We hypothesize that the ability of these acidic activators to specifically interact with multiple components of the transcription initiation complex likely underlies the dramatic functional synergy exhibited by this class of activation domains in vivo.
Asunto(s)
FN-kappa B/genética , Activación Transcripcional , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular , Proteínas de Unión al ADN/metabolismo , Electroquímica , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutagénesis Insercional , FN-kappa B/química , Oligopéptidos/química , Oligopéptidos/genética , Fenilalanina/química , Regiones Promotoras Genéticas , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteína de Unión a TATA-Box , Factor de Transcripción ReIA , Factor de Transcripción TFIIB , Factores de Transcripción/metabolismoRESUMEN
Human foamy virus encodes a nuclear regulatory protein, termed Bel-1, that serves as a potent activator of viral transcription. Mutational analysis has identified a small, discrete activation domain within Bel-1 that is highly active in both higher and lower eukaryotic cells. Here, we demonstrate that the activation domain of Bel-1 is highly dependent on the ADA2 transcriptional adaptor for biological activity in yeast cells, a property previously shown to be a hallmark of the VP16 class of acidic transcriptional activators (S. L. Berger, B. Pina, N. Silverman, G. A. Marcus, J. Agapite, J. L. Regier, S. J. Triezenberg, and L. Guarente, Cell 70:251-265, 1992). Using genetic selection in yeast cells, we have derived a set of point mutants within the Bel-1 activation domain that display a qualitatively similar loss in activation potential when examined in either yeast or human cells. These data indicate that the Bel-1 activation domain functions similarly in both lower and higher eukaryotes and strongly suggest that Bel-1 belongs to the VP16 class of acidic transcription factors.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de los Retroviridae/genética , Spumavirus/genética , Transactivadores/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Células HeLa , Proteína Vmw65 de Virus del Herpes Simple/genética , Humanos , Datos de Secuencia Molecular , Mutación Puntual , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/genética , Activación TranscripcionalRESUMEN
The formation of dimers or higher-order multimers is critical to the biological activity of many eukaryotic regulatory proteins. However, biochemical analyses of the multimerization capacity of the Tat trans activator of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) have yielded contradictory results. We used the two-hybrid genetic assay for protein-protein interactions in the eukaryote Saccharomyces cerevisiae (S. Fields and O.-K. Song, Nature [London] 340:245-246, 1989) to examine the multimerization of Tat in vivo. Both HIV-1 and HIV-2 Tat are shown to form specific homo- but not heteromultimers in the yeast cell nucleus. Mutational analysis indicates a critical role for the essential core motif of Tat in mediating this interaction but demonstrates that efficient Tat multimerization does not require an intact cysteine motif. These data raise the possibility that the multimerization of Tat may be important for Tat function in higher eukaryotes.
Asunto(s)
Núcleo Celular/metabolismo , Productos del Gen tat/metabolismo , VIH-1/metabolismo , VIH-2/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción , Cromosomas Fúngicos , Clonación Molecular/métodos , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Productos del Gen tat/genética , Sustancias Macromoleculares , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
The production of poliovirus capsid proteins from a capsid protein precursor (P1) is mediated by virus-encoded proteinase 3CD and involves a complicated set of proteinase-substrate interactions. In addition to substrate and enzymatic determinants required for this interaction, we describe a cellular cofactor, which facilitates 3CD recognition of the P1 precursor. Cellular cofactor activity is 3CD dependent and salt dependent. Our analysis shows that proteolytic cleavage of the P1 precursor at the VP0/VP3 cleavage site exhibits a greater dependency on the cellular cofactor than cleavage at the VP3/VP1 site. Such a greater dependency on cellular cofactor activity can be relieved (in part) by the substitution of an Ala residue for the Pro residue at the -4 position of the VP0/VP3 cleavage site. However, mutant viruses containing Pro-to-Ala substitutions at the -4 position of the VP0/VP3 site exhibit defects in viral growth.
Asunto(s)
Cápside/metabolismo , Cisteína Endopeptidasas/metabolismo , Endopeptidasas/metabolismo , Poliovirus/metabolismo , Proteasas Virales 3C , Secuencia de Aminoácidos , Clonación Molecular , Endopeptidasas/química , Células HeLa , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutación Puntual , Poliovirus/crecimiento & desarrollo , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Proteínas Virales/metabolismoRESUMEN
Intraoperative and postsurgical epidurally administered pain relief is associated with reduced morbidity. We reviewed the charts of 19 patients who had total esophagectomy to see whether the method of postoperative pain relief influenced the length of hospital stay and cost of the procedure. The patients received either intravenous (group M) or epidural (group E) morphine for postoperative pain. The length of stay in the intensive care unit was reduced by 2 1/2 days and total hospital stay by 7 days in the epidural group. This resulted in a saving of Canadian $12,770 per patient.
Asunto(s)
Esofagectomía , Dolor Postoperatorio/tratamiento farmacológico , Anciano , Analgesia Epidural , Costos y Análisis de Costo , Cuidados Críticos , Femenino , Humanos , Tiempo de Internación , Masculino , Persona de Mediana Edad , Morfina/administración & dosificación , Morfina/uso terapéutico , PremedicaciónRESUMEN
Research on the activity of self-cleaving proteases in bacterial, mammalian and virus-infected cells is reviewed, with an emphasis on the diversity of regulatory systems controlled by protein processing. Each of these three groups will be considered in turn by focusing on the following systems: the Rec A-dependent intramolecular cleavage of the Escherichia coli SOS response protein, LexA; the intramolecular activation of the mammalian aspartic acid protease, pepsinogen; and the autocatalytic cleavage of polyproteins synthesized by picornaviruses.
Asunto(s)
Péptido Hidrolasas/metabolismo , Procesamiento Proteico-Postraduccional , Serina Endopeptidasas , Proteínas Bacterianas/metabolismo , Modelos Biológicos , Pepsinógenos/metabolismo , Picornaviridae/metabolismo , Proteínas/metabolismo , Rec A Recombinasas/metabolismo , Proteínas Virales/metabolismoRESUMEN
Amino acid insertions or substitutions were introduced into the poliovirus P1 capsid precursor at locations proximal to the two known Q-G cleavage sites to examine the role of the P4 residue in substrate processing by proteinase 3CD. Analysis of the processing profile of P1 precursors containing four-amino-acid insertions into the carboxy terminus of VP3 or a single-amino-acid substitution at the P4 position of the VP3-VP1 cleavage site demonstrates that substitution of the alanine residue in the P4 position of the VP3-VP1 cleavage site significantly affects cleavage at that site by proteinase 3CD. A single-amino-acid substitution at the P4 position of the VP0-VP3 cleavage site, on the other hand, has only a slight effect on 3CD-mediated processing at this cleavage site. Finally, analysis of six amino acid insertion mutations containing Q-G amino acid pairs demonstrates that the in vitro and in vivo selection of a cleavage site from two adjacent Q-G amino acid pairs depends on the presence of an alanine in the P4 position of the cleaved site. Our data provide genetic and biochemical evidence that the alanine residue in the P4 position of the VP3-VP1 cleavage site is a required substrate determinant for the recognition and cleavage of that site by proteinase 3CD and suggest that the P4 alanine residue may be specifically recognized by proteinase 3CD.
Asunto(s)
Cápside/metabolismo , Cisteína Endopeptidasas/metabolismo , Genoma Viral , Poliovirus/enzimología , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Virales , Proteasas Virales 3C , Secuencia de Aminoácidos , Cápside/genética , ADN Viral/genética , Células HeLa , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Plásmidos , Poliovirus/genética , Precursores de Proteínas/genética , ARN Viral/genética , Mapeo Restrictivo , Especificidad por Sustrato , TransfecciónRESUMEN
A six-amino-acid insertion containing a Q-G amino acid pair was introduced into the carboxy terminus of the capsid protein VP3 (between residues 236 and 237). Transfection of monkey cells with full-length poliovirus cDNA containing the insertion described above yields a mutant virus (Sel-1C-02) in which cleavage occurs almost entirely at the inserted Q-G amino acid pair instead of at the wild-type VP3-VP1 cleavage site. Mutant Sel-1C-02 is delayed in the kinetics of virus production at 39 degrees C and exhibits a defect in VP0 cleavage into VP2 and VP4 at 39 degrees C. Sucrose gradient analysis of HeLa cell extracts prepared from cells infected by Sel-1C-02 at 39 degrees C shows an accumulation of fast-sedimenting replication-packaging complexes and a significant amount of uncleaved VP0 present in fractions containing mature virions. Our data provide in vivo evidence for the importance of determinants other than the conserved amino acid pair (Q-G) for recognition and cleavage of the P1 precursor by proteinase 3CD and show that an alteration in the carboxy terminus of VP3 or the amino terminus of VP1 affects the process of viral maturation.
Asunto(s)
Cápside/genética , Poliovirus/genética , Secuencia de Aminoácidos , Animales , Proteínas de la Cápside , Centrifugación por Gradiente de Densidad , Humanos , Metionina/metabolismo , Datos de Secuencia Molecular , Mutación , Mapeo Peptídico , Poliovirus/crecimiento & desarrollo , Procesamiento Proteico-Postraduccional/genética , ARN Viral/genéticaRESUMEN
A mutagenic oligonucleotide cassette was used to introduce single and tandem amino acid substitutions into the proteinase 3C coding region of an infectious poliovirus type 1 cDNA. The sites targeted for mutagenesis, residues 60, 61, and 66, are located within a putative helical loop structure which may be involved in substrate recognition by the enzyme. Fourteen viable 3C proteinase mutants were isolated. A Lys----Arg substitution at position 60 resulted in cold sensitivity for growth at 33 degrees. Replacement of Lys 60 with Ile, either singly or in combination with substitutions at position 61, resulted in viruses that produced three- to fivefold more 3D RNA polymerase than wild-type poliovirus. 3C-mediated processing of the remaining sites within the polyprotein was not noticeably affected. The overproduction of 3D is a consequence of more efficient processing of the carboxy-terminal Gln-Gly amino acid pair of 3C. Together with a previous report in which substitution of Val 54 with an Ala residue results in a poliovirus that produces decreased levels of 3D, these observations provide evidence that the putative loop region (residues 51-66) may be a functional domain involved in recognition of the carboxy-terminal Gln-Gly cleavage site of 3C.