THE VACCINIA VIRUS 14-KILODALTON (A27L) FUSION PROTEIN FORMS A TRIPLECOILED-COIL STRUCTURE AND INTERACTS WITH THE 21-KILODALTON (A17L) VIRUS MEMBRANE-PROTEIN THROUGH A C-TERMINAL ALPHA-HELIX

The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell an d cell-to-cell fusions. The protein is located on the surface of the i ntracellular mature virus form and is essential for both the release o f extracellular enveloped virus from the cells and virus spread. Seque nce analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 4 4 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linki ng studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, sup port the above-described structural division of the 14-kDa protein. Th e two contiguous cysteine residues at positions 71 and 72 are not resp onsible for the formation of 14-kDa protein trimers. The location of h ydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was support ed by the construction of a three-helix bundle model and molecular dyn amics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that do es not produce the 14-kDa protein during infection (VVindA27L) show th at the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted t hird or-helix on the C terminus of the 14-kDa protein, as the region i nvolved in protein binding. These findings were confirmed in vivo, fol lowing transfection of animal cells with plasmid vectors expressing mu tant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fu sion proteins, such as hemagglutinin of influenza virus and gp41 of hu man immunodeficiency virus, except for the presence of a protein-ancho ring domain instead of a transmembrane domain. Based on our observatio ns, we have established a structural model of the 14-kDa protein.