A cytoplasmic and transient viral expression vector which is replication incompetent would be very useful to study cellular functions in vitro or in vivo. Vesicular stomatitus virus (VSV) has an extremely high infectious unit to particle ratio with up to 1 in every 5 virus particles leading to infection. With HIV-1 this ratio is only 1:10000. The goal of this project is to generate defective VSV particles that can be used as transient gene expression vectors. VSV is a nonsegmented negative strand RNA virus and does not have a DNA intermediate. This is the reason why it is very difficult to obtain viral nucleocapsids which can be replicated. To generate these vectors, our detailed knowledge of the basic virology and pathogenesis of VSV will be used together with the latest recombinant DNA technology that now allows for the first time to generate VSV particles from recombinant DNA. To be able to generate a defective, helper virus-free SVS particle, we have cloned the large polymerase gene of VSV into several eukaryotic expression plasmids with different promoters and expression levels. These plasmids will require integration or they will exist as high copy number episomes in the nucleus. HEK 293 cells have recently been transfected with these DNAs and cell clones have been selected for drug resistance. These cells are currently being tested for their ability to complement temperature sensitive polymerase mutants as the restrictive temperature. Studies on whether helper virus functions could be provided by conditional VSV polymerase mutants were also carried out. We evaluated the potential of some of these mutants with respect to encapsidating complete VSV genomic RNA when it was transcribed by T7 RNA polymerase from plasmid DNA in tissue culture. As an alternative the nucleocapsid and polymerse proteins necessary for replication are provided by cotransfection of plasmid DNAs.
