Poxviruses, and in particular vaccinia virus, have been utilized as systems for the expression of proteins in eukaryotic cells and as vectors for antigen delivery. Their ability to incorporate large amounts of DNA and wide host range leads to the expression and correct processing of a great variety of proteins in many cell lines. High level expression vectors have been constructed by expressing bacteriophage RNA polymerase genes in vaccinia. These vectors express high levels of any gene located behind the bacteriophage promoter. We have developed a conditionally lethal vaccinia virus expression vector which expresses the T7 bacteriophage RNA polymerase. Under the restrictive conditions of elevated temperature, the virus undergoes an abortive infection where greatly reduced amounts of the vaccinia late or post-replicative genes are synthesized. We have quantitated the absolute level of expression of foreign genes in this system and compared its utility to presently available vaccinia T7 expression systems. Also, this vector provides a means for the analysis of potential trans-acting factors participating in vaccinia late processes such as resolution of DNA replicative intermediates. Poxvirus vectors are customarily constructed by introducing, the foreign DNA into the poxvirus genome by homologous recombination. An alternative method using direct ligation vectors has been developed by our lab to efficiently construct chimeric genomes in situations not readily amenable for homologous recombination. We have constructed and characterized direct ligation vectors engineered to contain restriction sites to fix the orientation of the insert DNA behind strongly expressing constitutive vaccinia promoters at the beginning of the thymidine kinase gene to utilize drug selection in the isolation of recombinants. These viruses provide a set of universally applicable, direct ligation, poxvirus cloning vectors, which extend the utility of poxvirus vectors for construction and expression of complex libraries. Our lab is currently working on the development of new poxvirus based vectors, including the development of direct ligation vectors for attenuated viruses with host range defects. These vectors will exhibit increased safety, especially for immunocompromised patients, since the infections will be self-limiting. One host-range, restricted-attenuated virus under consideration as a vector is modified virus ankara (MVA). As part of the characterization of MVA, our laboratory has participated in research aimed at determining the viral gene products missing in MVA which lead to the altered host range phenotype.
