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Development and characterization of herpes simplex virus type 1 vectors expressing m1 muscarinic acetylcholine receptors Atkinson, Leone Sheila


This work focuses primarily on the expression of a neurotransmitter receptor gene, the ml muscarinic acetylcholine receptor gene from HSV-1 recombinant viruses. The herpes simplex virus type-1 (HSV-1) has a number of biological features that make it an attractive vector for gene transfer into neurons: the virus is neurotropic, has a large genetic carrying capacity, produces high stock titers, and has the ability to establish latency in neurons. Viral-expressed m l receptors were characterized with regards to viral-associated effects on expression, pharmacological properties, and functional activity in tissue culture cells. HSV-1 recombinant viruses were made replication-defective by deletion of the a4 genes encoding the major regulatory protein ICP4. The elimination of ICP4 expression was found to enhance viral-directed ml receptor expression from these recombinant viruses. The 5' noncoding region of the ml receptor gene which contains three potential ICP4 repressor sites was shown to mediate ICP4 effects on viral-directed ml receptor expression. The viral host shutoff protein (vhs) was also eliminated in HSV-1 recombinants, and these recombinant viruses demonstrated reduced viral-associated effects on host polypeptide synthesis and slightly higher levels of viral-directed ml receptor expression. Virally expressed ml receptors maintained their pharmacological and functional properties. An HSV-1 recombinant virus defective in both ICP4 and vhs expression was used to analyze viral-directed ml receptor expression in primary cortical neuron cultures. The developmental age of the cultures influenced ml receptor expression and function in infected neurons. Infected ten-day-old cultures demonstrated maximum viraldirected ml receptor expression, and overexpression of ml receptors in these cultures produced an increase in functional response following muscarinic agoniststimulation. Investigations of ml receptor activities in neurons utilizing the HSV-1 recombinant virus were limited by cytopathic effects associated with viral infection and the lack of an available antibody against the ml receptor. In an effort to overcome these limitations, an HSV-1 amplicon vector expressing antigenic-tagged ml receptors was developed. This vector offered two advantages: firstly, the amplicon vector did not express any viral proteins, and therefore, avoided cytopathic effects associated with the recombinant viruses; and secondly, the attachment of an antigenic tag to the receptor enabled antibody recognition, and therefore, broadened the potential methods of examining viraldirected ml receptor expression. In addition, these studies enabled comparison of the advantages and disadvantages of HSV-1 recombinant viruses and HSV-1 amplicon vectors.

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