Title

A heat-inducible packaging cell line for simian virus 40

Date of Award

1990

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Walter A. Scott, Committee Chair

Abstract

To elucidate the role of DNA sequences in packaging of simian virus 40 (SV40) and to package recombinant DNA into SV40 pseudovirions two possible experimental approaches were explored. First, plasmids containing different SV40 DNA fragments were cotransfected with a plasmid which expresses structural proteins of SV40 but is too big to be packaged into pseudovirions. Second, permanent cell lines expressing genes for SV40 packaging proteins were constructed.Plasmids A to F were constructed by insertion of individual Sau3AI DNA fragments of SV40 into a vector containing an SV40 origin of replication. Each of plasmids was cotransfected into COS-7 cells (which contain integrated genes for SV40 T antigen and support replication of input DNA containing an SV40 origin of replication since COS-7 cells express large T antigen of SV40) together with a plasmid containing the entire SV40 genome uninterrupted in the packaging genes (the late genes of SV40 are VP1, VP2 and VP3). In these cotransfection experiments plasmids A-F were incorporated into infectious virus particles. Whether any specific DNA fragment of SV40 may facilitate packaging was not determined because of the appearance of unwanted recombinant DNA molecules in the cotransfected cells.The late genes of SV40 under heat shock promoter control were introduced into COS-7 cells to construct permanent cell lines which expressed the major capsid protein of SV40 (VP1). Isolation of these cell lines demonstrated that constitutive expression of VP1 could be tolerated by these cells. Viral DNA defective in the late region of SV40 was incorporated into virus particles using these cell lines; however, several experiments suggested the presence of unwanted recombinant DNA molecules, which could make these cells unsuitable for the study of the role of DNA sequences in virus assembly or for packaging of recombinant DNA into SV40 pseudovirions.Constitutive expression of large T antigen by COS-7 may favor recombination. Plasmid p114 was constructed in which both early and late genes of SV40 were expressed under control of the heat shock regulatory elements. Following introduction of p114 into BSC-40 cells (monkey kidney cells which can tolerate higher temperatures) one colony derived from a single cell was isolated (designated 114F cell line). This cell line expressed large T antigen, VP1, VP2 and VP3 under heat shock control. 114F cells supported heat shock dependent replication of input DNA containing an SV40 origin of replication. Plasmids A-F were packaged in heat shocked 114F cells although plasmids containing different SV40 sequences were packaged with different efficiencies. The sedimentation properties of pseudovirions A-F were determined and they were shown to be DNase I resistant and free of recombinant genomes. This cell line may be useful for efficient packaging of recombinant DNA into SV40 pseudovirions.

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

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