The project fosuses on the development of a potentially highly efficient gene expression vector based on vesicular stomatitis virus (VSV). To be able to employ defective VSV particles, we have attempted to isolate cell clones expressing the VSV polymerase protein. We have isolated several different cell lines that either constitutively or upon induction express the viral polymerase protein L, a 241 kDa protein. After infection of these cell lines at the non-permissive temperature with temperature sensitive polymerase mutants, we observed typical cytopathic effects such as cell rounding which was not observed with parental cell lines. Pathogenesis indicated that complementation of the mutant virus occurred in all cells. Spread of the temperature sensitive virus, however, did not occur. It has been reported that temperature sensitive VSV mutants can severely interfere with wild type virus replication similar to defective interfering virus particles. The long-range goal is to generate defective VSV viruses encoding foreign genes but lacking the large polymerase gene itself. We anticipate that a helper cell line providing polymerase L protein may be able to support a productive infection and the replication of a defective particle in contrast to a temperature sensitive mutant. We have isolated a DNA construct that encodes the VSV genome with most of the polymerase gene deleted. Since VSV shuts off cellular transcription and/or mRNA transport from the nucleus through a function of the viral matrix protein, the matrix gene was replaced by a mutant matrix gene that does not shut off the cell. We have obtained DNA clones encoding the viral nucleocapsid protein N, the phosphoprotein P and polymerase L. These DNAs will in the future be co-expressed with the defective VSV DNA construct. A recombinant vaccinia virus will be used initially to provide T7 RNA polymerase for precise expression of all four DNAs. The efficiency to generate VSV particles through co-transfection of DNA has been shown to be very low. A single ribonucleocapsid formed under these conditions, however, may be sufficient to sustain an infection in cells that constitutively express the polymerase L. The generation and helper virus free replication of a recombinant defective VSV particle will be essential to create this novel expression system for the study of cellular functions.
