Poxviruses are large, enveloped, double-stranded DNA viruses that replicate in the cytoplasm of host cells and are responsible for diseases of humans and other animals. Vaccinia virus (VACV), the most extensively studied member in the family, encodes approximately 200 proteins, of which 100 are conserved in all members of the vertebrate subfamily of poxviruses and have roles in gene expression, DNA replication, morphogenesis and cell entry. Previous studies have shown that several vaccinia virus proteins localize to the endoplasmic reticulum (ER), suggesting that it serves as the source of viral membranes. Determining the topology of these viral proteins can provide information about protein function and viral membrane formation. My first project involved using an asymmetric self-associating split-GFP system to determine the topology of the transmembrane viral proteins L2 and A30.5 that localize in the endoplasmic reticulum. This split-GFP system uses large (215 aa) and small (16 aa) fragments of GFP that fluoresce only upon complementation. Our results showed that a short GFP fragment can be used to tag small transmembrane viral proteins to determine their localization and topology in vivo. The second project focuses on a protein called I2, which I showed is required for later stage virion morphogenesis. I deleted the I2 gene from the VACV genome by homologous recombination. The I2-deletion mutant was unable to replicate in control cells demonstrating that the protein has an essential role in VACV replication. Transmission electron microscopy revealed a striking defect in virus morphogenesis. During normal VACV morphogenesis, spherical immature particles shed the viral D13 scaffold protein and assume the brick shape of mature infectious particles. However, this transition did not occur in cells infected with the I2-deletion mutant and dense spherical particles accumulated. Furthermore, the scaffold protein was retained on the defective particles. In addition, the levels of membrane proteins comprising the entry/fusion complex were greatly diminished in these particles although most other proteins were present at normal levels. Based on these data, I2 is important for removal of the scaffold protein from immature virus particles, which is necessary for subsequent steps in morphogenesis including the incorporation of the entry/fusion proteins into the viral membrane.