| Vaccinia virus (VV), the prototypic poxvirus, is closely related to smallpox virus and was the vaccine utilized in the successful WHO campaign to eradicate smallpox. Vaccinia remains in use as a vaccine, but also serves as the focus of most experimental research into poxvirus biology. Vaccinia was the first virus to be visualized by microscopy, grown in tissue culture and purified. In recent years, the use of inducible and temperature sensitive (ts) mutant viruses has led to substantial advances in the poxvirus field, including the identification and understanding of the viral proteins required for replication and transcription of the viral genome and the complex process of virion morphogenesis.; The viral membrane protein A14 plays an essential role in viral morphogenesis. Repression of A14, which results in a 1000-fold reduction in viral yield, leads to an early block in viral morphogenesis characterized by the accumulation of large virosomes, empty "crescents" which fail to contact these virosomes, and most strikingly, large numbers of aberrant 25-nm vesicles. Here we report the establishment of a transient complementation system for the structure/function analysis of A14.; The vaccinia virus proteins A30 and G7 are known to play essential roles in early morphogenesis, acting prior to the formation of immature virions. Their repression or inactivation results in the accumulation of large virosomes, detached membrane crescents and empty immature virions. We have undertaken further study of these proteins to place them within the context of the F10 kinase, the A14 membrane protein, and the H5 phosphoprotein, which have been the focus of previous studies within our laboratory. Here we confirm that both A30 and G7 undergo F10 kinase-dependent phosphorylation in vivo , and recapitulate the modification of A30 in vitro. Although the detached crescents observed upon loss of A30 or G7 echo those seen upon repression of A14, no interaction between A30/G7 and A14 could be detected. We did, however, determine that the A30 and G7 proteins are unstable during non-permissive tsH5 infections, suggesting that the loss of A30/G7 contributes to the phenotype of tsH5 infections. (Abstract shortened by UMI.)... |
