Vaccinia virus (VV) encodes at least six proteins that are modified by the addition of a 14-carbon saturated fatty acid through an amide linkage and at least eight proteins that are modified post-translationally by the addition of a 16-carbon saturated fatty acid through linkage to cysteine residues. These post-translational modifications are referred to as myristylation and palmitylation, respectively. The purpose of this work was to further characterize the known myristylproteins and to define a consensus motif for the palmitylation of a protein so that we could identify and begin the characterization of new palmitylproteins. Through this work we have identified a loosely conserved consensus motif that directs the palmitylation of a protein. Using the VV palmitylprotein p37, we characterized this motif and then used it in the identification three new VV palmitylproteins. We have also determined the membrane orientation of the VV myristylproteins, L1R, within the intracellular mature virus (IMV) particle. Hydrophobicity plot analysis identified two possible membrane orientations based on two putative transmembrane domains. Through transient expression data, L1R was determined to span the IMV membrane twice, with both the amino and carboxy termini being on the lumen side. Three lac recombinant viruses which are inducible for the A16L, E7R, and G9R open reading frames were created and analyzed using a newly developed vector system that fuses the green fluorescent protein to the neomycin resistance gene. Propagation of these viruses in the absence of the inducer IPTG determined that these genes are essential to VV replication. We have identified and characterized the primary structural determinants specifying the modification of a protein by palmitate, and have identified three new VV palmitylproteins. In addition, the membrane orientation of the VV myristylprotein L1R was deduced, which can enable the construction of better recombinant vaccines through efficient antigen presentation. Lastly, we have developed a technique and vector system to easily create and isolate VV recombinants for characterization. This system enabled us to further characterize the A16L, E7R, and G9R myristylproteins
