RESUMEN
A sensitive and quantitative cell-free infection assay, utilizing recombinant human T-cell leukemia virus type 1 (HTLV-1)-based vectors, was developed in order to analyze early events in the virus replication cycle. Previous difficulties with the low infectivity and restricted expression of the virus have prevented a clear understanding of these events. Virus stocks were generated by transfecting cells with three plasmids: (i) a packaging plasmid encoding HTLV-1 structural and regulatory proteins, (ii) an HTLV-1 transfer vector containing either firefly luciferase or enhanced yellow fluorescent protein genes, and (iii) an envelope expression plasmid. Single-round infections were initiated by exposing target cells to filtered supernatants and quantified by assaying for luciferase activity in cell extracts or by enumerating transduced cells by flow cytometry. Transduction was dependent on reverse transcription and integration of the recombinant virus genome, as shown by the effects of the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) and by mutation of the integrase gene in the packaging vector, respectively. The 50% inhibitory concentration of AZT was determined to be 30 nM in this HTLV-1 replication system. The stability of HTLV-1 particles, pseudotyped with either vesicular stomatitis virus G protein or HTLV-1 envelope, was typical of retroviruses, exhibiting a half-life of approximately 3.5 h at 37 degrees C. The specific infectivity of recombinant HTLV-1 virions was at least 3 orders of magnitude lower than that of analogous HIV-1 particles, though both were pseudotyped with the same envelope. Thus, the low infectivity of HTLV-1 is determined in large part by properties of the core particle and by the efficiency of postentry processes.
Asunto(s)
Vectores Genéticos , Virus Linfotrópico T Tipo 1 Humano/fisiología , Recombinación Genética , Replicación Viral , Línea Celular , Línea Celular Transformada , Sistema Libre de Células , Genes Reporteros , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Luciferasas/genética , Transcripción Genética , Células Tumorales Cultivadas , Ensamble de Virus , Integración ViralRESUMEN
Prenatal providers are reluctant to discuss alcohol use in the clinical setting, even though heavy alcohol use is associated with fetal alcohol syndrome (FAS) and fetal alcohol effects (FAE), sometimes known as alcohol-related neurodevelopmental disorder. Fourteen percent to 20% of pregnant women report drinking some alcohol during pregnancy. Approximately 0.2% to 1% meet the criteria for heavy drinking. Reducing drinking during pregnancy has the potential to reduce the risk for FAS and FAE. Routine screening for alcohol use during pregnancy followed by referrals for those considered to be at risk is recommended. Women are often more receptive to intervention during pregnancy, as they focus on positive health behaviors. A number of brief screening tools designed for use on a routine basis are reviewed. Physicians who learn to comfortably discuss alcohol use during pregnancy can help substantially reduce the impact of these disorders.
Asunto(s)
Consumo de Bebidas Alcohólicas/efectos adversos , Trastornos del Espectro Alcohólico Fetal/prevención & control , Tamizaje Masivo , Complicaciones del Embarazo/prevención & control , Adulto , Femenino , Conductas Relacionadas con la Salud , Humanos , Relaciones Médico-Paciente , Embarazo , Atención PrenatalRESUMEN
The visna virus Tat protein is required for efficient viral transcription from the visna virus long terminal repeat (LTR). AP-1 sites within the visna virus LTR, which can be bound by the cellular transcription factors Fos and Jun, are also necessary for Tat-mediated transcriptional activation. A potential mechanism by which the visna virus Tat protein could target the viral promoter is by protein-protein interactions with Fos and/or Jun bound to AP-1 sites in the visna virus LTR. Once targeted to the visna virus promoter, the Tat protein could then interact with basal transcription factors to activate transcription. To examine protein-protein interactions with cellular proteins at the visna virus promoter, we used an in vitro protein affinity chromatography assay and electrophoretic mobility shift assay, in addition to an in vivo two-hybrid assay, to show that the visna virus Tat protein specifically interacts with the cellular transcription factors Fos and Jun and the basal transcription factor TBP (TATA binding protein). The Tat domain responsible for interactions with Fos and Jun was localized to an alpha-helical domain within amino acids 34 to 69 of the protein. The TBP binding domain was localized to amino acids 1 to 38 of Tat, a region previously described by our laboratory as the visna virus Tat activation domain. The bZIP domains of Fos and Jun were found to be important for the interactions with Tat. Mutations within the basic domains of Fos and Jun abrogated binding to Tat in the in vitro assays. The visna virus Tat protein was also able to interact with covalently cross-linked Fos and Jun dimers. Thus, the visna virus Tat protein appears to target AP-1 sites in the viral promoter in a mechanism similar to the interaction of human T-cell leukemia virus type 1 Tax with the cellular transcription factor CREB, by binding the basic domains of an intact bZIP dimer. The association between Tat, Fos, and Jun would position Tat proximal to the viral TATA box, where the visna virus Tat activation domain could contact TBP to activate viral transcription.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Productos del Gen tat/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Secuencias Repetidas Terminales , Factores de Transcripción/metabolismo , Virus Visna-Maedi/genética , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Sitios de Unión , Factores de Unión a la G-Box , Humanos , Ovinos , Proteína de Unión a TATA-BoxRESUMEN
The visna virus Tat protein is a strong transcriptional activator and is necessary for efficient viral replication. The Tat protein regulates transcription through an AP-1 site proximal to the TATA box within the viral long terminal repeat (LTR). Previous studies from our laboratory using Tat-Gal4 chimeric proteins showed that Tat has a potent acidic activation domain. Furthermore, a region adjacent to the Tat activation domain contains a highly conserved leucine-rich domain which, in the context of the full-length protein, suppressed the activity of the activation domain. To further elucidate the role of this region, four leucine residues within this region of Tat were mutated. In transient-transfection assays using visna virus LTR-CAT as a reporter construct, the activity of this leucine mutant was dramatically reduced. Additionally, domain-swapping experiments using the N-terminal activation domain of VP16 showed that the leucine-rich domain of Tat confers AP-1 responsiveness to the chimeric VP16-Tat protein. A chimeric VP16-Tat construct containing the leucine mutations showed no increased AP-1 responsiveness in comparison with that of the VP16 activation domain alone. Furthermore, in competition experiments, a Gal4-Tat protein containing only the leucine region of Tat (amino acids 34 to 62) was able to inhibit by competition the activity of full-length Tat. These studies strongly suggest that this leucine-rich domain is responsible for targeting the Tat protein to AP-1 sites in the viral LTR. In addition, examination of the amino acid sequence of this region of Tat revealed a highly helical secondary structure and a pattern of residues similar to that in the leucine zippers in the bZIP family of DNA-binding proteins. This has important implications for the interaction of Tat with cellular proteins, specifically Fos and Jun, that contain bZIP domains.
Asunto(s)
ADN Viral/metabolismo , Productos del Gen tat/metabolismo , Leucina/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Factor de Transcripción AP-1/metabolismo , Virus Visna-Maedi/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Cartilla de ADN , Células HeLa , Proteína Vmw65 de Virus del Herpes Simple/genética , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación Puntual , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ovinos , Relación Estructura-Actividad , Virus Visna-Maedi/genéticaRESUMEN
OBJECTIVE: To determine the knowledge, clinical experience and perceived needs for resource materials of Saskatchewan physicians in regard to fetal alcohol syndrome (FAS) and alcohol-related birth defects. DESIGN: Mailed survey. SETTING: Saskatchewan. PARTICIPANTS: All 48 pediatricians and half (394) of the family physicians (FPs) and general practitioners (GPs) practising in Saskatchewan received a questionnaire. The numbers of physicians who completed it were 24 and 249 respectively. RESULTS: The pediatricians were more likely than the other physicians to be aware of FAS and to have diagnosed at least one case of FAS. Among the FPs and GPs, the year of graduation from medical school was a significant factor in their knowledge of FAS and their diagnostic practices. Those who graduated before 1974, the year FAS was first described in the medical literature, were less likely than the more recent graduates to be aware of FAS and to ask their patients about alcohol use during pregnancy but were more likely to feel comfortable discussing alcohol-related issues in families. All of the groups reported a need for more information about FAS and for resources on alcohol-related issues in general. CONCLUSIONS: Saskatchewan physicians are aware of FAS but have expressed a need for more information about FAS, particularly for parents, as well as physician training materials and information about where to refer patients with FAS and parents with alcohol-related problems.
Asunto(s)
Trastornos del Espectro Alcohólico Fetal/diagnóstico , Conocimientos, Actitudes y Práctica en Salud , Pediatría , Médicos de Familia , Adulto , Anciano , Humanos , Recién Nacido , Persona de Mediana Edad , Pediatría/educación , Médicos de Familia/educación , Médicos de Familia/psicología , Saskatchewan , Encuestas y CuestionariosRESUMEN
Visna virus is a pathogenic lentivirus of sheep tat is distantly related to the primate lentiviruses, including human immunodeficiency virus type 1. The visna virus genome encodes a small regulatory protein, Tat, which is necessary for efficient viral replication and enhanced viral transcription. To investigate the mechanism of action of the visna Tat protein and to localize the protein domain(s) responsible for transcriptional activation, chimeric proteins containing visna virus Tat sequences fused to the DNA binding domain of the yeast transactivation factor GAL4 (residues 1 to 147) were made. The GAL4-Tat fusion proteins were transfected into cells and tested for the ability to activate the adenovirus E1b promoter via upstream GAL4 DNA binding sites. Full-length GAL4-Tat fusion proteins were weak transactivators in this system, giving only a two- to fourfold increase in transcription in several cell types, including HeLa and sheep choroid plexus cells. In contrast, fusion of the N-terminal region of the Tat protein to GAL4 revealed a potent activation domain. Amino acids 13 to 38 appeared to be the most critical for activation. No other region of the protein showed any activation in the GAL4 system. This N-terminal region of the visna virus Tat protein has a large number of acidic and hydrophobic residues, suggesting that Tat has an acidic activation domain common to many transcriptional transactivators. Mutations in hydrophobic and bulky aromatic residues dramatically reduced the activity of the chimeric protein. Competition experiments suggest that mechanism of the visna virus Tat activation domain may closely resemble that of the herpesvirus activator VP16 and human immunodeficiency virus Tat, a related lentivirus activator, since both significantly reduce the level of visna virus Tat activation. Finally, a domain between residues 39 and 53 was identified in the Tat protein that, in the GAL4 system, negatively regulates activation by Tat.
Asunto(s)
Productos del Gen tat/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción , Activación Transcripcional , Virus Visna-Maedi/metabolismo , Proteínas E1B de Adenovirus/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/biosíntesis , Plexo Coroideo/metabolismo , Plexo Coroideo/microbiología , Cartilla de ADN , Proteínas de Unión al ADN , Proteínas Fúngicas/biosíntesis , Productos del Gen tat/biosíntesis , VIH-1/metabolismo , Células HeLa , Herpesviridae/metabolismo , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Primates , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Ovinos , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Since the identification of fetal alcohol syndrome (FAS) in 1973, significant inroads have been made towards understanding the effects of alcohol on fetal development. However, it is not clear if these findings are considered clinically relevant by pediatricians. This survey was designed to assess clinical knowledge, practice, and attitudes concerning alcohol-related birth defects. Data were collected in a questionnaire that was mailed to 234 randomly selected Massachusetts pediatricians. Responses suggest that a substantial proportion of pediatricians have knowledge about the effects of alcohol on pregnancy. However, many considered themselves unprepared to deal with this topic. More physicians suspected FAS/FAE than made the diagnosis. Almost three fourths reported they would find professional education in this area helpful. Broader dissemination of research findings in clinically relevant formats and improving the sense of preparedness among pediatricians have the potential to improve the care of children born to heavily drinking pregnant women.
Asunto(s)
Actitud del Personal de Salud , Educación Médica Continua , Trastornos del Espectro Alcohólico Fetal/diagnóstico , Pediatría/educación , Femenino , Trastornos del Espectro Alcohólico Fetal/prevención & control , Humanos , Recién Nacido , Massachusetts , Relaciones Médico-Paciente , EmbarazoRESUMEN
Consumption of alcohol during pregnancy is well recognized as a risk factor associated with adverse fetal development. While precise safe or dangerous levels of maternal drinking have not been identified, it is clear that the women who drink most heavily are at the greatest risk. Prevention of alcohol-related birth defects requires development of programs directed to the special needs of addicted women and their families. The nature of addiction suggests that direct interventions focused on changing individual drinking behavior have the best chance of success.