Asunto(s)

Complejo SIDA Demencia/virología , Barrera Hematoencefálica , Sistema Nervioso Central/fisiología , Sistema Nervioso Central/virología , VIH/fisiología , Complejo SIDA Demencia/sangre , Animales , Sistema Nervioso Central/irrigación sanguínea , Técnicas de Cultivo , Modelos Animales de Enfermedad , Endotelio Vascular/citología , Endotelio Vascular/virología , Humanos

RESUMEN

The UL4 gene of herpes simplex virus type 1 is predicted to encode a 21.5 kDa protein of 199 amino acids. Although UL4 is dispensable for growth in cell culture, its function is not known. In the present study, the promoter of UL4 was examined and found to contain a cAMP-response element which bound the transcription factor CREB, and was strongly activated by cAMP. A recombinant virus, termed UL4HS, was constructed with a nonsense linker inserted into the UL4 open reading frame, to make a truncated UL4 protein of 60 amino acids. In addition, a marker-rescued virus, UL4HSMR, was constructed. Western immunoblot analysis revealed a 23 kDa band in extracts of wild-type and marker-rescued virus infected cells which was missing for UL4HS. Only modest differences were observed in the abilities of wild-type and UL4-mutant viruses to grow in Vero cells or in contact-inhibited mouse C3H/10T1/2 cells. In addition, there were only modest differences between the ability of UL4HS to replicate in murine corneas and trigeminal ganglia relative to wild-type viruses, and reactivation of UL4HS from latency was unaffected. Taken together, these data demonstrate that UL4 is dispensable for latency and pathogenesis in mice.

Asunto(s)

Herpesvirus Humano 1/fisiología , Proteínas Virales/genética , Latencia del Virus , Secuencia de Aminoácidos , Animales , Células Cultivadas , Chlorocebus aethiops , AMP Cíclico/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/crecimiento & desarrollo , Humanos , Cinética , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Mutagénesis , Células PC12 , Regiones Promotoras Genéticas , Ratas , Recombinación Genética , Células Vero , Proteínas Virales/fisiología , Activación Viral , Replicación Viral

RESUMEN

A neuroinvasive/neuropathogenic SIV variant termed SIVmac182 was previously isolated and characterized (Watry et al, 1994). This neuroinvasive strain was derived from the uncloned strain SIVmac251 through serial animal passage of infected microglia, unlike previously reported neurovirulent strains. Importantly, the virus described here was isolated from a strain which already demonstrates limited neuroinvasiveness in vivo, through a route of inoculation which exerts selective pressure for variants in the periphery that can naturally cross the blood-brain barrier and gain access to the brain. Examination of animal tissues indicated that the neuroinvasive strain was capable of replicating in brain microvascular endothelial cells (BMEC). Therefore, we developed an in vitro model of BMEC infection in which to examine mechanisms of virus neuroinvasiveness and neuropathogenicity as well as to address mechanisms of HIV-induced dementia. Results obtained with this in vitro system indicate that growth in BMEC may predict neuroinvasiveness in vivo, and furthermore, that brain passage of virus results in the generation of neuroinvasive strains which demonstrate an increased efficiency of BMEC infection in vitro.

Asunto(s)

Endotelio Vascular/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Complejo SIDA Demencia/virología , Animales , Antígenos Virales/análisis , Linfocitos B/citología , Linfocitos B/virología , Encéfalo/irrigación sanguínea , Encéfalo/virología , Separación Celular , Clonación Molecular , ADN Viral/análisis , Modelos Animales de Enfermedad , Encefalitis Viral/virología , Técnica del Anticuerpo Fluorescente , Productos del Gen gag/análisis , Humanos , Células Híbridas/citología , Células Híbridas/virología , Hibridación in Situ , Macaca mulatta , Virus de la Inmunodeficiencia de los Simios/clasificación , Virus de la Inmunodeficiencia de los Simios/crecimiento & desarrollo , Linfocitos T/citología , Linfocitos T/virología , Virulencia

RESUMEN

The herpes simplex virus type 1 virion host shutoff protein has four domains whose sequences are conserved only among neurotropic herpesviruses. Mutant viruses with 29- and 31-amino-acid deletions in domains III and IV but outside of the domain required for interaction with VP16 were generated. The mutants failed to induce cellular RNA degradation and showed impaired virulence in mice. Domains III and IV are therefore required for both shutoff and virulence.

Asunto(s)

Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Herpes Simple/virología , ARN Viral/metabolismo , Simplexvirus/metabolismo , Proteínas Virales/genética , Animales , Chlorocebus aethiops , Ratones , Mutación , ARN Viral/genética , Simplexvirus/genética , Simplexvirus/patogenicidad , Células Vero , Proteínas Virales/metabolismo , Virulencia

RESUMEN

The herpes simplex virus type 1 (HSV-1) UL41 gene product, virion host shutoff (vhs), has homologs among five alphaherpesviruses (HSV-1, HSV-2, pseudorabies virus, varicella-zoster virus, and equine herpesvirus 1), suggesting a role for this protein in neurotropism. A mutant virus, termed UL41NHB, which carries a nonsense linker in the UL41 open reading frame at amino acid position 238 was generated. UL41NHB and a marker-rescued virus, UL41NHB-R, were characterized in vitro and tested for their ability to replicate in vitro and in vivo and to establish and reactivate from latency in a mouse eye model. As demonstrated by Western blotting (immunoblotting) and Northern (RNA) blotting procedures, UL41NHB encodes an appropriately truncated vhs protein and, as expected for a vhs null mutant, fails to induce the degradation of cellular glyceraldehyde-3-phosphate dehydrogenase mRNA. The growth of UL41NHB was not significantly altered in one-step growth curves in Vero or mouse C3H/10T1/2 cells but was impaired in corneas, in trigeminal ganglia, and in brains of mice compared with the growth of KOS and UL41NHB-R. As a measure of establishment of latency, quantitative DNA PCR showed that the amount of viral DNA within trigeminal ganglia latently infected with UL41NHB was reduced by approximately 30-fold compared with that in KOS-infected ganglia and by 50-fold compared with that in UL41NHB-R-infected ganglia. Explant cocultivation studies revealed a low reactivation frequency for UL41NHB (1 of 28 ganglia, or 4%) compared with that for KOS (56 of 76, or 74%) or UL41NHB-R (13 of 20 or 65%). Taken together, these results demonstrate that vhs represents a determinant of viral pathogenesis.

Asunto(s)

Encéfalo/virología , Herpes Simple/patología , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/patogenicidad , Ganglio del Trigémino/virología , Proteínas Virales/fisiología , Latencia del Virus , Animales , Secuencia de Bases , Encéfalo/patología , Línea Celular , Chlorocebus aethiops , Cartilla de ADN , ADN Viral/análisis , Femenino , Herpesvirus Humano 1/genética , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos , Datos de Secuencia Molecular , Mutagénesis Insercional , Reacción en Cadena de la Polimerasa , Ribonucleasas , Ganglio del Trigémino/patología , Células Vero , Proteínas Virales/biosíntesis , Virión/genética , Virión/patogenicidad , Virión/fisiología

RESUMEN

The promoters of the latency-associated transcripts (LATs) of herpes simplex virus type 1 (HSV-1) strains KOS and McKrae were compared to examine their influence upon the reactivation phenotypes of these two strains. Unlike strain KOS, McKrae is readily reactivable using in vivo reactivation models. We found greater than 96% sequence conservation between KOS and McKrae in the LATs promoter region, and both promoters showed equivalent basal and inducible activities. An inter-strain recombinant (termed MK13) was constructed in which the LATs promoter of HSV-1 McKrae was recombined into the background of HSV-1 strain KOS. In a murine u.v. light-induced reactivation model, virus shedding was detected by eye swabbing in two of 44 (5%) mice infected with KOS, 20 of 42 (48%) mice infected with McKrae and none of 45 (0%) mice infected with MK13. These data show that the LATs promoters of these viruses are structurally and functionally similar and that transfer of the LATs promoter from McKrae into KOS is insufficient to confer a reactivatable phenotype.

Asunto(s)

Herpesvirus Humano 1/genética , Queratitis Herpética/microbiología , Regiones Promotoras Genéticas , Transcripción Genética , Replicación Viral , Animales , Secuencia de Bases , Bucladesina/farmacología , Cloranfenicol O-Acetiltransferasa/biosíntesis , Colforsina/farmacología , Secuencia Conservada , Biblioteca Genómica , Herpesvirus Humano 1/patogenicidad , Herpesvirus Humano 1/fisiología , Ratones , Datos de Secuencia Molecular , Células PC12 , Recombinación Genética , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Transfección

RESUMEN

A mutant allele of the maize brittle-1 (bt1) locus, brittle-1-mutable (bt1-m), was shown genetically and molecularly to result from the insertion of a defective Suppressor-mutator (dSpm) transposable element. An Spm-hybridizing restriction enzyme fragment, which cosegregates with the bt1-m allele and is absent from wild-type revertants of bt1-m, was identified and cloned. Non-Spm portions of it were used as probes to identify wild-type (Bt1) cDNAs in an endosperm library. The 4.3-kb bt1-m genomic clone contains a 3.3-kb dSpm, which is inserted in an exon and is composed of Spm termini flanking non-Spm sequences. RNA gel blot analyses, using a cloned Bt1 cDNA probe, indicated that Bt1 mRNA is present in the endosperm of developing kernels and is absent from embryo or leaf tissues. Several transcripts are produced by bt1-m. The deduced translation product from a 1.7-kb Bt1 cDNA clone has an apparent plastid transit peptide at its amino terminus and sequence similarity to several mitochondrial inner-envelope translocator proteins, suggesting a possible role in amyloplast membrane transport.

Asunto(s)

Proteínas de la Membrana/genética , Proteínas de Plantas/genética , Supresión Genética , Zea mays/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN , Elementos Transponibles de ADN , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Fenotipo , Proteínas de Plantas/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

Starch gel electrophoresis of extracts from developing maize (Zea mays L.) endosperms 22 days postpollination reveals only a single zone of phosphoglucomutase activity in the majority of the inbreds tested. The other inbreds had the expected two zones of activity. The activity that is present in all inbreds is the amyloplast isozyme while the absent form is a cytosolic enzyme. The lack of the cytosolic isozyme has no discernible phenotypic consequences.