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1.
Antiviral Res ; 52(3): 251-9, 2001 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11675142

RESUMEN

The novel influenza virus neuraminidase (NA) inhibitor, (1S,2S,3R,4R)-3-[(1S)-(acetylamino)-2-ethylbutyl]-4-[(aminoiminomethyl)amino]-2-hydroxy-cyclopentanecarboxylic acid (RWJ-270201, BCX-1812), is a potent inhibitor of influenza A and B viruses in cell culture and in infected mice. A mouse-adapted strain of influenza A/Shangdong/09/93 (H3N2) virus was serially passaged in the presence of 1 microM compound. After the fourth passage, breakthrough of resistant virus occurred. By the tenth passage, a twice plaque purified isolate was obtained which could replicate in 10 microM inhibitor. The 50% effective concentration (EC(50)) values for RWJ-270201 against wild-type and resistant viruses, determined by using a cytopathic effect inhibition assay, were 0.007 and 23 microM, respectively. Cross-resistance to zanamivir and oseltamivir carboxylate was observed. The hemagglutinin (HA) and NA genes of the virus were sequenced to determine the mutation(s) which conferred drug resistance. No differences were found between the resistant and wild-type viruses in the NA gene. However, a point mutation resulting in a single amino acid change (Lys189Glu) was found in the resistant viral HA. The wild-type and resistant viruses were compared for virulence in BALB/c mice. The resistant virus was approximately tenfold less virulent than the wild-type virus based upon virus challenge dose. Mice infected with a lethal dose of the resistant virus could still be effectively treated with RWJ-270201. Thus, the HA mutation may allow for the spread of the virus in cell culture in the presence of the NA inhibitor, but not in mice.


Asunto(s)
Antivirales/farmacología , Ciclopentanos/farmacología , Farmacorresistencia Viral , Subtipo H3N2 del Virus de la Influenza A , Virus de la Influenza A/enzimología , Virus de la Influenza A/genética , Neuraminidasa/antagonistas & inhibidores , Ácidos Carbocíclicos , Animales , Antivirales/química , Antivirales/uso terapéutico , Línea Celular , Línea Celular Transformada , Ciclopentanos/química , Ciclopentanos/uso terapéutico , Perros , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Guanidinas , Humanos , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/patogenicidad , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Ratones , Ratones Endogámicos BALB C , Mutación/genética , Virulencia/genética
2.
J Immunol ; 163(9): 4960-9, 1999 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-10528200

RESUMEN

Positive selection of thymocytes is a complex and crucial event in T cell development that is characterized by cell death rescue, commitment toward the helper or cytotoxic lineage, and functional maturation of thymocytes bearing an appropriate TCR. To search for novel genes involved in this process, we compared gene expression patterns in positively selected thymocytes and their immediate progenitors in mice using the differential display technique. This approach lead to the identification of a novel gene, mIAN-1 (murine immune-associated nucleotide-1), that is switched on upon positive selection and predominantly expressed in the lymphoid system. We show that mIAN-1 encodes a 42-kDa protein sharing sequence homology with the pathogen-induced plant protein aig1 and that it defines a novel family of at least three putative GTP-binding proteins. Analysis of protein expression at various stages of thymocyte development links mIAN-1 to CD3-mediated selection events, suggesting that it represents a key player of thymocyte development and that it participates to peripheral specific immune responses. The evolutionary conservation of the IAN family provides a unique example of a plant pathogen response gene conserved in animals.


Asunto(s)
Secuencia Conservada , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Genes de Plantas , Familia de Multigenes/inmunología , Timo/citología , Timo/inmunología , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Biomarcadores/química , Complejo CD3/fisiología , Diferenciación Celular/inmunología , Clonación Molecular , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Linfocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Técnicas de Cultivo de Órganos , Especificidad de Órganos/genética , Proteínas de Plantas/genética , ARN Mensajero/biosíntesis , Homología de Secuencia de Aminoácido , Transducción de Señal/genética , Transducción de Señal/inmunología , Timo/química , Timo/metabolismo
3.
J Neurovirol ; 2(6): 388-96, 1996 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-8972420

RESUMEN

FIV is a lentivirus of domestic cats that causes a spectrum of diseases that is remarkably similar to the clinical syndrome produced by HIV infection in people. Both HIV and FIV has been shown to cause neurologic dysfunction. Specific Pathogen-Free (SPF) cats were placed into one of three groups: FIV-PPR infected; DU-FIV-PPR (a dUTPase mutant of the FIV-PPR clone) infected; or an age-matched control group. In both infected groups, the general clinical signs of infection included lymphadenopathy, oral ulcerations, rough hair coat, and conjuntivitis. Specific neurological changes in the FIV-PPR infected cats included hind limb paresis; delayed righting and pupillary reflexes; behavioral changes; delayed visual and auditory evoked potentials; decreased spinal and peripheral nerve conduction velocities; and marked alterations in sleep patterns. Most of these changes were also observed in the DU-FIV-PPR infected cats. However, these cats tended to have a slightly less severe disease. In this study, we have demonstrated that an infectious molecular clone of FIV closely parallels the disease course of wild type FIV-infected cats. By using a knockout gene mutant of this clone, we were able to demonstrate that the dUTPase gene is not essential for neuropathogenesis. Further use of the FIV-PPR clone should prove useful in determining the essential viral elements that are important in the neuropathogenesis of lentiviral infections.


Asunto(s)
Síndrome de Inmunodeficiencia Adquirida del Felino/fisiopatología , Virus de la Inmunodeficiencia Felina/genética , Enfermedades del Sistema Nervioso/virología , Animales , Gatos , Clonación Molecular , Electroencefalografía , Potenciales Evocados Auditivos del Tronco Encefálico , Potenciales Evocados Somatosensoriales , Síndrome de Inmunodeficiencia Adquirida del Felino/virología , Genes Virales/genética , Virus de la Inmunodeficiencia Felina/patogenicidad , Mutagénesis/fisiología , Enfermedades del Sistema Nervioso/fisiopatología , Conducción Nerviosa/fisiología , Nervios Periféricos/enzimología , Nervios Periféricos/fisiopatología , Nervios Periféricos/virología , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Sueño/fisiología , Médula Espinal/enzimología , Médula Espinal/fisiopatología , Médula Espinal/virología , Virulencia
4.
Nat Biotechnol ; 14(13): 1685-91, 1996 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-9634852

RESUMEN

Differential gene expression occurs in the process of development, maintenance, injury, and death of unicellular as well as complex organisms. Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Electronic subtraction (ES), subtractive hybridization (SH), and differential display (DD) are methods commonly used for this purpose. A rigorous examination has been lacking and therefore quantitative aspects of these methods remain speculative. We compare these methods by identifying a total of 58 unique differentially expressed mRNAs within the same experimental system (HeLa cells treated with interferon-gamma). ES yields digital, reusable data that quantitated steady-state mRNA concentrations but only identified abundant mRNAs (seven were identified), which represent a small fraction of the total number of differentially expressed mRNAs. SH and DD identified abundant and rare mRNAs (33 and 23 unique mRNAs respectively) with redundancy. The redundancy is mRNA abundance-dependent for SH and primer-dependent for DD. We conclude that DD is the method of choice because it identifies mRNAs independent of prevalence, uses small amounts of RNA, identifies increases and decreases of mRNA steady-state levels simultaneously, and has rapid output.


Asunto(s)
Clonación Molecular/métodos , Expresión Génica/genética , ARN Mensajero/biosíntesis , Antivirales/farmacología , Northern Blotting , Recuento de Células , ADN Complementario/genética , Biblioteca de Genes , Células HeLa , Humanos , Interferón gamma/farmacología , Hibridación de Ácido Nucleico/métodos , ARN Mensajero/análisis , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética
5.
Proc Natl Acad Sci U S A ; 92(16): 7480-4, 1995 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-7638216

RESUMEN

Feline immunodeficiency virus (FIV) encodes the enzyme deoxyuridine-triphosphatase (DU; EC 3.6.1.23) between the coding regions for reverse transcriptase and integrase in the pol gene. Here, we report the in vivo infection of cats with a DU- variant of the PPR strain of FIV and compare its growth properties and tissue distribution with those of wild-type FIV-PPR. The results reveal several important points: (i) DU- FIV is able to infect the cat, with kinetics similar to that observed with wild-type FIV; (ii) both wild-type and DU- FIV-infected specific-pathogen free cats mount a strong humoral antibody response which is able to limit the virus burden in both groups of animals; (iii) the virus burden is reduced in the DU- FIV-infected cats, particularly in tissues such as spleen and salivary gland; and (iv) the mutation frequency in DU- FIVs integrated in the DNA of primary macrophages after 9 months of infection is approximately 5-fold greater than the frequency observed in DU- FIV DNA integrated in T lymphocytes. Mutation rate with wild-type FIV remains the same in both cell types in vivo. The dominant mutations seen in macrophages with DU- FIV are G-->A base changes, consistent with an increased misincorporation of deoxyuridine into viral DNA of DU- FIVs during reverse transcription. Because this enzyme is absent from human immunodeficiency virus type 1 and other primate lentiviruses, virus replication in cell environments with low DU activity may lead to increased mutation and contribute to the rapid expansion of the viral repertoire.


Asunto(s)
Virus de la Inmunodeficiencia Felina/enzimología , Virus de la Inmunodeficiencia Felina/genética , Mutación , Pirofosfatasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cápside/genética , Cápside/inmunología , Gatos , Cartilla de ADN/genética , ADN Viral/genética , Síndrome de Inmunodeficiencia Adquirida del Felino/virología , Genes pol , Genoma Viral , Virus de la Inmunodeficiencia Felina/crecimiento & desarrollo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Distribución Tisular
6.
Virology ; 196(2): 451-7, 1993 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-8396797

RESUMEN

The feline lentivirus, FIV, contains dUTPase (DU) as part of the enzyme cassette encoded by the viral pol gene (Elder et al., 1992, J. Virol. 66, 1791-1794). The enzyme is processed from the Pol polyprotein and is packaged into infectious virions. We report here the basic characteristics of the viral enzyme, including substrate specificity, ion requirements, and pH optimum. We also report the overexpression of DU in Escherichia coli and insertional mutagenesis of the enzyme in the context of the complete provirus or DU alone. The enzyme requires Mg2+ for full activity and competition studies employing unlabeled dNTPs indicated that DU has an absolute preference for dUTP. The pH optimum for FIV DU is pH 7.0. The limits of the protein dictate a species of M(r) 14,350, which agrees precisely with the determination by ion spray mass spectroscopy of DU isolated from virions. Cleavage sites at the junctions between DU, RT, and IN, as defined by N-terminal amino acid sequencing of each protein species, are consistent with predictions for sites of cleavage by aspartate protease. In-frame insertional mutagenesis at Tyr 75 of DU abolishes activity. Cells transfected with proviruses containing this mutation express virion-associated reverse transcriptase activity but lack DU activity. The resultant virions replicate slower than those possessing wild-type DU. Tests are currently underway to evaluate the consequences of DU mutagenesis on in vivo phenotype.


Asunto(s)
Virus de la Inmunodeficiencia Felina/enzimología , Pirofosfatasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Gatos , Células Cultivadas , Clonación Molecular , Análisis Mutacional de ADN , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Virus de la Inmunodeficiencia Felina/genética , Magnesio/farmacología , Datos de Secuencia Molecular , Mutagénesis Insercional , Pirofosfatasas/biosíntesis , Pirofosfatasas/efectos de los fármacos , Proteínas Recombinantes/biosíntesis , Homología de Secuencia de Aminoácido , Especificidad por Sustrato
7.
J Virol ; 67(4): 1869-76, 1993 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-8383214

RESUMEN

N-terminal amino acid sequencing, ion spray mass spectrometry, and cleavage of synthetic peptide substrates were used to identify the N and C termini of the mature Gag and Pol proteins of feline immunodeficiency virus (FIV). The Gag polyprotein encodes matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. The Gag-Pol polyprotein encodes, in addition to the above proteins, protease (PR), reverse transcriptase (RT), dUTPase (DU), and integrase (IN). Secondary cleavage of RT at Trp-595-Tyr-596 of Pol yields a truncated form lacking the C-terminal RNase H domain. The observed and expected molecular masses of the viral proteins were in agreement, with three exceptions. (i) The molecular mass of MA was 14,735 Da, compared with a predicted mass of 14,649 Da, based on a single cleavage at Tyr-135-Pro-136 of Gag. The observed molecular mass is consistent with myristoylation of MA, which was confirmed by metabolic labeling of FIV MA with [3H]myristic acid. (ii) The N terminus of the NC protein is generated via cleavage at Gln-366-Val-367 of Gag, which predicts a mass of 25,523 for CA and 9,101 for the major form of NC. The observed mass of CA was 24,569, consistent with loss of nine C-terminal amino acids by a second cleavage of CA at Leu-357-Leu-358. Synthetic FIV protease accurately cleaved synthetic peptide substrates containing this site. (iii) The actual mass of NC (7,120 Da) was approximately 2 kDa smaller than the mass predicted by synthesis to the stop codon at the end of Gag (9,101 Da). Experiments are in progress to characterize additional cleavage(s) in NC.


Asunto(s)
Productos del Gen gag/metabolismo , Productos del Gen pol/metabolismo , Virus de la Inmunodeficiencia Felina/metabolismo , Secuencia de Aminoácidos , Western Blotting , Endopeptidasas/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Ácido Mirístico , Ácidos Mirísticos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/química
8.
J Clin Microbiol ; 27(5): 832-6, 1989 May.
Artículo en Inglés | MEDLINE | ID: mdl-2745694

RESUMEN

A variety of chemically defined compounds were tested to characterize the substrate specificity of the influenza C virus esterase and to determine whether a substrate could be found that would be useful in an assay to detect the virus. Two new substrates, alpha-naphthyl acetate and alpha-naphthyl propionate, were identified; alpha-naphthyl acetate was employed to develop an assay specific for influenza type C virus in MDCK cells. The assay was sufficiently sensitive to detect esterase activity in a single cell and distinguished influenza C virus infections from those of types A and B viruses. Infected cells could be detected as early as 8 h postinfection, with maximal enzyme detection occurring at 24 h. Assay of influenza C virus in the chorioallantoic or amniotic fluid of infected eggs was performed by applying fluids directly onto nitrocellulose strips and then incubating with alpha-naphthyl acetate. Both the cellular and nitrocellulose-bound assays are rapid, inexpensive, and easy to perform, offering advantages for use in clinical laboratories.


Asunto(s)
Esterasas/metabolismo , Gammainfluenzavirus/aislamiento & purificación , Naftalenos/metabolismo , Orthomyxoviridae/aislamiento & purificación , Propionatos/metabolismo , Animales , Embrión de Pollo , Concentración de Iones de Hidrógeno , Hidrólisis , Gammainfluenzavirus/enzimología , Naftoles/metabolismo , Nitrofenoles/metabolismo , Especificidad por Sustrato , Temperatura
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