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K Neary

Publications and source records attributed to K Neary.

8 recordsLinked to original sources
PubMed1991

Protease sensitivity and nuclease resistance of the scrapie agent propagated in vitro in neuroblastoma cells.

The scrapie agent has been propagated in vitro in mouse neuroblastoma cells. To further characterize the tissue culture-derived scrapie agent, we studied the effects of protease and nuclease digestion on the agent derived from these cells. The scrapie agent in these cells was found to be resistant to protease digestions for short times but was inactivated by prolonged digestion at high protease concentrations. In contrast, digestion with a variety of nucleases did not alter the agent titer. These results demonstrate that the agent requires an essential protein or proteins for infectivity. If the agent also contains a nucleic acid genome, it must be more nuclease resistant than the majority of cellular DNA and RNA. These properties of the tissue culture-derived scrapie agent were identical to those of brain-derived scrapie agent and thus cannot be attributed to secondary effects of tissue pathology, since the infected cell cultures show no cytopathic effects as a result of infection.

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PubMed1990

Bovine papillomavirus E2 repressor mutant displays a high-copy-number phenotype and enhanced transforming activity.

The methionine codon at bovine papillomavirus type 1 nucleotide 3091 was mutated to determine whether it may serve as an initiation codon for an E2 transcriptional repressor protein and to determine the role of the repressor in the biological activities of the virus. A series of transient expression experiments with CV1 cells documented that the mutation reduced expression of repressor activity from the viral genome and resulted in increased expression of the E5 transforming gene. Viral genomes containing the mutation displayed enhanced transforming activity in several assays in mouse C127 cells, including focus formation, colony formation in agarose, and tumorigenicity. In transformed cells, the mutant viral DNA was maintained as a plasmid with approximately 500 genomes per cell, whereas the wild-type copy number was approximately 75. These results indicate that the wild-type bovine papillomavirus type 1 genome encodes an E2 repressor protein that moderates the viral transforming activity and allows maintenance of the viral DNA at a relatively low copy number.

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PubMed1990

Normal and scrapie-associated forms of prion protein differ in their sensitivities to phospholipase and proteases in intact neuroblastoma cells.

Previous studies have indicated that scrapie infection results in the accumulation of a proteinase K-resistant form of an endogenous brain protein generally referred to as prion protein (PrP). The molecular nature of the scrapie-associated modification of PrP accounting for proteinase K resistance is not known. As an approach to understanding the cellular events associated with the PrP modification in brain tissue, we sought to identify proteinase K-resistant PrP (PrP-res) in scrapie-infected neuroblastoma cells in vitro and to compare properties of PrP-res with those of its normal proteinase K-sensitive homolog, PrP-sen. PrP-res was detected by immunoblot in scrapie-infected but not uninfected neuroblastoma clones. Densitometry of immunoblots indicated that there was two- to threefold more PrP-res than PrP-sen in one infected clone. Metabolic labeling and membrane immunofluorescence experiments indicated that PrP-sen was located on the cell surface and could be removed from intact cells by phosphatidylinositol-specific phospholipase C and proteases. In contrast, PrP-res was not removed after reaction with these enzymes. Thus, either the scrapie-associated PrP-res was not on the cell surface or it was there in a form that is resistant to these hydrolytic enzymes. Attempts to detect intracellular PrP-res by immunofluorescent staining of fixed and permeabilized cells revealed that PrP was present in discrete perinuclear Golgi-like structures. However, the staining pattern was similar in both scrapie-infected and uninfected clones, and thus the intracellular staining may have represented only PrP-sen. Analysis of scrapie infectivity in cells treated with extracellular phospholipase, proteinase K, and trypsin indicated that, like PrP-res, the scrapie agent was not removed from the infected cells by any of these enzymes.

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PubMed1989

Papillomavirus-associated inductions of cellular proteins in mouse C127 cells: correlation with the presence of open reading frame E2.

Bovine papillomavirus type 1 (BPV-1) readily transforms mouse C127 cells, conferring the ability to grow in soft agar and to form tumors in athymic (nu/nu) mice. Electrophoresis of total cellular proteins from these BPV-transformed lines on ultra-high resolution, giant two-dimensional gels displays the presence of novel, papillomavirus-related protein phenotypes. Analysis of the established BPV-1-transformed C127 cell lines, ID13 and ID14, reveals a set of six proteins which are either absent or synthesized at extremely low levels in the parental cell line. One of these proteins is also present in v-ras-transformed C127 cells, but none of the others are found in cells transformed by a variety of viral oncogenes, including the polyomavirus middle T, v-mos, or v-fes. The genome of BPV-1 contains two separate open reading frames (ORFs), E5 and E6, which can act independently to transform C127 cells. In addition, trans-activator and repressor proteins encoded respectively by the full-length and carboxy-terminal E2 ORF regulate the level of expression of other BPV-1 genes. We examined 34 cell lines transformed by intact and subgenomic recombinant DNAs of BPV-1. Cells harboring BPV-1 DNAs engineered to eliminate the expression of ORFs E4, E5, E6, or E7 display five of the PV-associated proteins, but these proteins are not seen in lines lacking the full E2 ORF. Moreover, G418-selected nontransformed cells expressing E2 cDNA from an SV40 promoter exhibit these proteins at high levels. Surprisingly, these proteins are also present in cells containing BPV-1 DNAs with amino-terminal E2 deletions, suggesting that these PV-associated proteins represent novel cellular responses to a factor encoded within the E2-C gene region.

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PubMed1989

Open reading frames E6 and E7 of bovine papillomavirus type 1 are both required for full transformation of mouse C127 cells.

A series of mutations in open reading frames (ORFs) E6 and E7 of bovine papillomavirus type 1 (BPV1) was constructed to analyze the roles of these ORFs in transformation of mouse C127 cells. The mutations were designed to prevent synthesis of specific proteins encoded by these genes. None of the mutations caused a decrease in the focus-forming activity of the full-length viral genome or in the ability of the viral DNA to replicate as a high-copy-number plasmid. Analysis of these mutants in the absence of a functional BPV1 E5 gene revealed a weak focus-forming activity encoded by ORF E6. Mutations preventing synthesis of the E6 protein did cause defects in anchorage-independent growth and tumorigenicity of transfected and transformed cells. However, a frameshift mutation between the first and second ATG codons of ORF E6 did not inhibit induction of colony formation, suggesting that translation from the first methionine codon is not required. Mutations that inactivated ORF E7 or E6/E7 individually did not inhibit induction of colony formation in agarose. However, a defect in this activity was caused by simultaneous disruption of both ORF E7 and ORF E6/E7 when they were expressed from the full-length viral genome but not when they were expressed under the control of a retrovirus long terminal repeat. These results suggest that translation of both ORF E6 and the 3' end of ORF E7 is required for efficient induction of anchorage-independent growth by the intact BPV1 genome.

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PubMed1987

Mutational analysis of open reading frame E4 of bovine papillomavirus type 1.

Open reading frame (ORF) E4 is a 353-base-pair ORF of bovine papillomavirus type 1. To determine the biological activities of this ORF in mouse C127 cells, we analyzed the effects of two constructed mutations which are predicted to prevent synthesis of ORF E4 proteins while leaving the amino acid sequence encoded by the overlapping ORF E2 unchanged. Neither mutation interfered with the abilities of the mutants to efficiently induce focus formation, induce growth in soft agarose, or transactivate an inducible bovine papillomavirus type 1 enhancer. Also, neither mutation prevented establishment of the viral DNA as an extrachromosomal plasmid in transformed cells. These results suggest that ORF E4 proteins are not required for these biological activities, and they are consistent with the observation of others (J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986) that the ORF E4 protein of a human papillomavirus is associated with late gene expression during papilloma formation.

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