Assessing the Structure and Function of Vaccinia Virus Gene Products by Transient Complementation.

Abstract

Poxviruses are large, complex dsDNA viruses that are highly unusual in replicating solely within the cytoplasm of the infected cell. The most infamous poxvirus was variola virus, the etiological agent of smallpox; today, poxviruses remain of biomedical significance, both as pathogens and as recombinant vaccines and oncolytic therapies. Vaccinia virus is the prototypic poxvirus for experimental analysis. The 195kb dsDNA genome contains >200 genes that encode proteins involved in such processes as viral entry, gene expression, genome replication and maturation, virion assembly, virion egress, and immune evasion.Molecular genetic analysis has been instrumental in the study of the structure and function of many viral gene products. Temperature-sensitive (ts) mutants have been especially useful in this endeavor; inducible recombinants and deletion mutants are now also important tools. Once a phenotype is observed following the repression, deletion, or inactivation of a particular gene product, the technique of transient complementation becomes central for further study.Simply put, transient complementation involves the transient expression of a variety of alleles of a given viral gene within infected cells, and the evaluation of which of these alleles can "complement" or "rescue" the phenotype caused by the loss of the endogenous allele. This analysis leads to the identification of key domains, motifs, and sites of posttranslational modification. Subcellular localization and protein:protein interactions can also be evaluated in these studies. The development of a reliable toolbox of vectors encoding viral promoters of different temporal classes, and the use of a variety of epitope tags, has greatly enhanced the utility of this experimental approach for poxvirus research.