Phenotypic consequences of rearranging the P, M, and G genes of vesicular stomatitis virus

The nonsegmented negative-strand RNA viruses (order Mononegavirales) includ e many important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene express ion, since genes that are close to the single promoter site at the 3' end o f the viral genome are transcribed at higher levels than those that occupy more distal positions. We manipulated an infectious cDNA clone of the proto typic vesicular stomatitis virus (VSV) to rearrange three of the five viral genes, using an approach which left the viral nucleotide sequence otherwis e unaltered. The central three genes in the gene order, which encode the ph osphoprotein P, the matrix protein M, and the glycoprotein G, were rearrang ed into all six possible orders. Viable viruses were recovered from each of the rearranged cDNAs. The recovered viruses were examined for their levels of gene expression, growth potential in cell culture, and virulence in mic e. Gene rearrangement changed the expression levels of the encoded proteins in concordance with their distance from the 3' promoter. Some of the virus es with rearranged genomes replicated as well or slightly better than wildt ype virus in cultured cells, while others showed decreased replication. All of the viruses were lethal for mice, although the time to symptoms and dea th following inoculation varied. These data show that despite the highly co nserved gene order of the Mononegavirales, gene rearrangement is not lethal or necessarily even detrimental to the virus. These findings suggest that the conservation of the gene order observed among the Mononegavirales may r esult from immobilization of the ancestral gene order due to the lack of a mechanism for homologous recombination in this group of viruses. As a conse quence, gene rearrangement should be irreversible and provide an approach f or constructing viruses with novel phenotypes.