Biomedical subjects

Deborah Schuback

Publications and source records attributed to Deborah Schuback.

3 recordsLinked to original sources

PubMed2004

HSV-1 virions engineered for specific binding to cell surface receptors.

Expression of specific peptide epitopes on the surface of virions has significant potential for studying viral biology and designing vectors for targeted gene therapy. In this study, an HSV-1 amplicon plasmid expressing a modified glycoprotein C (gC), in which the heparan sulfate binding domain was replaced with a His-tag, was used in generating HSV-1 virions. Western blot analysis demonstrated the presence of modified gC in the purified virions. The amplicon vectors were packaged using a gC-, lacZ+ helper virus to generate a mixture of high-titer helper virus (lacZ+) and amplicon vectors (GFP+), which expressed modified gC in the virion envelope. His-tagged virions bound to 293 6H cells expressing a cell surface pseudo-His-tag receptor four-fold more efficiently than to parental 293 cells and also proved more effective than wild-type virus in binding to both cell types. Binding resulted in productive infection by the modified virions with expression of reporter genes and cytopathic effect comparable to those of wild-type virions. Thus, not only can HSV-1 tropism be manipulated to recognize a non-herpes simplex binding receptor, but it is also possible to increase the infective capacity of the vectors beyond that of the wild-type virus via specific ligand receptor combinations.

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PubMed2003

TorsinA in PC12 cells: localization in the endoplasmic reticulum and response to stress.

Most cases of early-onset torsion dystonia are caused by deletion of GAG in the coding region of the DYT1 gene encoding torsinA. This autosomal dominant neurologic disorder is characterized by abnormal movements, believed to originate from neuronal dysfunction in the basal ganglia of the human brain. The torsins (torsinA and torsinB) are members of the "ATPases associated with a variety of cellular activities" (AAA(+)) superfamily of proteins that mediate chaperone and other functions involved in conformational modeling of proteins, protection from stress, and targeting of proteins to cellular organelles. In this study, the intracellular localization and levels of endogenous torsin were evaluated in rat pheochromocytoma PC12 cells following differentiation and stress. TorsinA, apparent MW 37 kDa, cofractionates with markers for the microsomal/endoplasmic reticulum (ER) compartment and appears to reside primarily within the ER lumen based on protease resistance. TorsinA immunoreactivity colocalizes with the lumenal ER protein protein disulfide isomerase (PDI) and extends throughout neurites. Levels of torsinA did not increase notably in response to nerve growth factor-induced differentiation. None of the stress conditions tested, including heat shock and the unfolded protein response, affected torsinA, except for oxidative stress, which resulted in an increase in the apparent MW of torsinA and redistribution to protrusions from the cell surface. These findings are consistent with a relatively rapid covalent modification of torsinA in response to oxidative stress causing a change in state. Mutant torsinA may interfere with and/or compromise ER functions, especially in dopaminergic neurons, which have high levels of torsinA and are intrinsically vulnerable to oxidative stress.

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PubMed2003

HSV-1 amplicon peptide display vector.

There are significant uses for expressing foreign peptide epitopes in viral surface attachment proteins in terms of investigating viral targeting, biology, and immunology. HSV-1 attachment, followed by fusion and entry, is mediated in large part by the binding of viral surface glycoproteins to cell surface receptors, primarily through heparan sulfate (HS) glycosaminoglycan residues. We constructed a HSV-1 amplicon plasmid (pCONGA) carrying the gC primary attachment protein gene with unique restriction sites flanking the HS binding domain (HSBD) (residues 33-176) to allow rapid, high efficiency substitution with foreign peptide domains. To test this system, a His tag with an additional unique restriction site (for selection and assay digests) was recombined into the pCONGA HSBD site to create pCONGAH. Infection of pCONGAH transfected Vero cells with HSV-1 helper virus (gCdelta2-3 or hrR3) produced His-modified gC as demonstrated by western blot analysis with co-localization of anti-gC and anti-His tag antibodies to a protein of appropriate molecular weight (50 kd). As CONGA and CONGAH amplicons carry a GFP transgene and the gCdelta2-3 and hrR3 viruses carry a lacZ transgene, vector stocks produced from 1 x 10(5) Vero cells could be titered for competent vector on cell monolayers and were demonstrated to contain 2 x 10(5) amplicon vector transducing units (t.u.)/ml and 1 x 10(7) virus t.u./ml. As the amplicon plasmids also contain the neomycin resistance gene (neo(r)), long term vector producer cell lines were created using G418 selection. This amplicon system provides means to rapidly and efficiently generate HSV-1 amplicon and viral vector expressing surface attachment proteins modified with different peptide epitopes for investigational and therapeutic uses, with the advantages of an amplicon plasmid that can be used with interchangeable helper virus vectors, is designed specifically for easy manipulation, and carries GFP and neo(r) transgenes for marker and selection functions.

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