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Biomedical subjects

F Tufaro

Publications and source records attributed to F Tufaro.

46 records · Page 3Linked to original sources
PubMed1991

Brefeldin A arrests the maturation and egress of herpes simplex virus particles during infection.

Herpes simplex virus (HSV) requires the host cell secretory apparatus for transport and processing of membrane glycoproteins during the course of virus assembly. Brefeldin A (BFA) has been reported to induce retrograde movement of molecules from the Golgi to the endoplasmic reticulum and to cause disassembly of the Golgi complex. We examined the effects of BFA on propagation of HSV type 1. Release of virions into the extracellular medium was blocked by as little as 0.3 microgram of BFA per ml when present from 2 h postinfection. Characterization of infected cells revealed that BFA inhibited infectious viral particle formation without affecting nucleocapsid formation. Electron microscopic analyses of BFA-treated and untreated cells (as in control cells) demonstrated that viral particles were enveloped at the inner nuclear membrane in BFA-treated cells and accumulated aberrantly in this region. Most of the progeny virus particles observed in the cytoplasm of control cells, but not that of BFA-treated cells, were enveloped and contained within membrane vesicles, whereas many unenveloped nucleocapsids were detected in the cytoplasm of BFA-treated cells. This suggests that BFA prevents the transport of enveloped particles from the perinuclear space to the cytoplasmic vesicles. These findings indicate that BFA-induced retrograde movement of molecules from the Golgi complex to the endoplasmic reticulum early in infection arrests the ability of host cells to support maturation and egress of enveloped viral particles. Furthermore, we demonstrate that the effects of BFA on HSV propagation are not fully reversible, indicating that maturation and egress of HSV type 1 particles relies on a series of events which cannot be easily reconstituted after the block to secretion is relieved.

Animals↗
PubMed1990

Herpes simplex virus particles are unable to traverse the secretory pathway in the mouse L-cell mutant gro29.

The mouse L-cell mutant gro29 was selected for its ability to survive infection by herpes simplex virus type 1 (HSV-1) and is defective in the propagation of HSV-1 and vesicular stomatitis virus (F. Tufaro, M. D. Snider, and S. L. McKnight, J. Cell Biol. 105:647-657, 1987). In this report, we show that gro29 cells harbor a lesion that inhibits the egress of HSV-1 virions during infection. We also found that HSV-1 glycoprotein D was slow to traverse the secretory pathway en route to the plasma membrane of infected gro29 cells. The movement of glycoproteins was not blocked entirely, however, and immunofluorescence experiments revealed that infected gro29 cells contained roughly 10% of the expected amount of glycoprotein D on their cell surface at 12 h postinfection. Furthermore, nucleocapsids and virions assembled inside the cells during infection, suggesting that the lesion in gro29 cells impinged on a late step in virion maturation. Electron micrographs of infected cells revealed that many of the intracellular virions were contained in irregular cytoplasmic vacuoles, similar to those that accumulate in HSV-1-infected cells treated with the ionophore monensin. We conclude from these results that gro29 harbors a defect that blocks the egress of HSV-1 virions from the infected cell without seriously impeding the flux of individual glycoproteins to the cell surface. We infer that HSV-1 maturation and egress require a host cell component that is either reduced or absent in gro29 cells and that this lesion, although not lethal to the host cell, cannot be tolerated by HSV-1 during its life cycle.

Animals↗
PubMed1989

Serological distance coefficients.

We define experimentally measurable similarity coefficients S[A,B/C] that specify the extent to which a pair of substances, A and B, are similar in the context of a diverse reagent, C, which can be a non-immune serum. We describe how this definition can be applied to mixtures of antibodies, for example the antibodies of two serum samples, A and B. A "distance coefficient" D[A,B/C] between two sera, A and B, in the context of C, is defined as one minus the similarity coefficient of the two sera in the same context. We discuss the problem of the experimental measurement of these coefficients, and the possible use of the coefficients in the diagnosis and prognosis of disease conditions.

AIDS Serodiagnosis↗
PubMed1987

Identification and characterization of a mouse cell mutant defective in the intracellular transport of glycoproteins.

We have isolated a mutant line of mouse L cells, termed gro29, in which the growth of herpes simplex virus (HSV) and vesicular stomatitis virus (VSV) is defective. The block occurs late in the infectious cycle of both viruses. We demonstrate that HSV and VSV enter gro29 cells normally, negotiate the early stages of infection, yet are impaired at a late stage of virus maturation. During VSV infection of the mutant cell line, intracellular transport of its glycoprotein (G protein) is slowed. Pulse-chase experiments showed that oligosaccharide processing is impeded, and immunofluorescence localization revealed an accumulation of G protein in a juxtanuclear region that contains the Golgi complex. We conclude that export of newly made glycoproteins is defective in gro29 cells, and speculate that this defect may reflect a lesion in the glycoprotein transport apparatus.

Animals↗
PubMed1987

Post-transcriptional restriction of gene expression in sea urchin interspecies hybrid embryos.

The synthesis of many paternal species-specific proteins is reduced in all stages of sea urchin interspecies hybrid embryos, due to the reduced amounts of some paternal mRNA species in hybrid embryos compared with the embryos of the paternal species (Tufaro and Brandhorst 1982). Possible explanations for this restriction were tested. Cloned cDNAs were selected that were specific for paternal RNA sequences having reduced amounts (to 2-20% of normal) in hybrid embryos derived from a cross of Stronglyocentrotus purpuratus eggs with Lytechinus pictus sperm. Several of these RNA species are barely detectable in the eggs, but they accumulate extensively (5- to 40-fold) during L. pictus embryogenesis. Thus, the restricted expression of these paternal genes in hybrid embryos is not the result of the persistence of stable maternal mRNA species stored in eggs and not replaced by zygotic transcription. The accumulation of some of these L. pictus transcripts is also reduced in the reciprocal cross (L. pictus eggs X S. purpuratus sperm); therefore, the full expression of these L. pictus genes in hybrid embryos is not dependent on species-specific maternal factors stored in the egg. The transcriptional activity of one such gene was estimated using a run-on assay in isolated nuclei; it is as actively transcribed in hybrid as it is in homospecific embryos, but in hybrid embryos the cytoplasmic transcript accumulates to only 2-15% of the normal level. Sequence analysis indicates that this gene encodes a metallothionein. Mechanisms are discussed that might account for the post-transcriptional restriction of expression of some genes in hybrid embryos.

Animals↗
PubMedDate not supplied

Herpes simplex virus: discovering the link between heparan sulphate and hereditary bone tumours.

To gain entry into the host, viruses use host cell surface molecules that normally serve as receptors for other ligands. Herpes simplex virus type 1 (HSV-1) uses heparan sulphate (HS) glycosaminoglycans (GAGs) as receptors for initial attachment to the host cell surface. HS GAGs are both ubiquitous and structurally diverse, and normally serve as critical mediators of interactions between the cell and the extracellular environment. We have used the HS binding ability of HSV-1 to identify the function of a cellular gene, EXT1, which is involved in HS polymerisation. Cellular factors that affect virus growth and replication are often key regulators of the cell cycle and EXT1 is no different-humans with inherited mutations in EXT1 have developmental defects that lead to bone tumours (hereditary multiple exostoses, HME) and sometimes chondrosarcomas. Thus, as a result of using HSV-1 as a molecular probe, a functionally orphaned disease gene now has a defined function. These findings highlight the utility of viruses for investigating important cellular processes.

Cell Line↗
PubMedDate not supplied

Development of a model to demonstrate photosensitizer-mediated viral inactivation in blood.

A model has been developed to demonstrate the use of photodynamic treatment (PDT) to eradicate viral contaminants from donated blood and blood products. Whole blood, spiked with vesicular stomatitis virus (VSV), was treated with the photosensitizer benzoporphyrin derivative-monoacid ring A (BPD-MA). After light activation of BPD-MA, a neutral red dye uptake assay was carried out to determine virus inactivation. Various drug incubation times and light intensities were tested as well as red cell lysis and distribution of VSV in blood. At BPD-MA concentrations between 2 and 4 micrograms per mL in whole blood, up to 10(7) VSV were inactivated. Several photosensitizers were also tested with this model to determine their relative efficacy in viral inactivation.

Blood↗
PubMedDate not supplied

Toxin resistance and reduced secretion in a mouse L-cell mutant defective in herpes virus propagation.

The mouse L-cell mutant gro29 was selected originally for its inability to propagate herpes simplex virus; it shows severe defects in virus egress and the transport and processing of viral glycoproteins after infection. In this report, we show that uninfected gro29 cells display pleiotropic changes in protein secretion, oligosaccharide processing, and sensitivity to the toxins ricin and modeccin. Specifically, the rate of secretion of a nonglycosylated protein, human growth hormone, was reduced 70% in gro29 cells compared with the parental L cells. A direct measurement of the transport capacity of Golgi membranes in a cell-free assay suggests that gro29 cells contain less functional Golgi than parental cells. Despite this deficiency, N-linked oligosaccharides were processed efficiently in mutant cells, although there were differences in the structure of the mature forms. Lectin intoxication assays revealed that gro29 cells were cross-resistant to killing by the cytotoxic lectins ricin and modeccin, but not to wheat germ agglutinin, Ricinus communis agglutinin RCA120, or leucoagglutinin. Fluorescence labeling using fluorescein-conjugated lectins showed that uninfected gro29 cells expressed relatively few ricin-binding molecules, suggesting a possible mechanism for toxin resistance. These studies provide evidence that the processes of protein secretion, lectin intoxication, and herpes virus maturation and egress may share a common cellular component.

Animals↗