The identification and characterization of a monoclonal antibody to the vaccinia virus E3 protein

Monoclonal antibodies with reactivity to vaccinia virus specific proteins are useful reagents to study the proteins as well as to help understand aspects of the poxvirus life cycle. Using a vaccinia virus proteomics microarray, we found a hybridoma (MAb 3015B2) from a vaccinia virus vaccinated mouse that reacted with the product of the E3L gene. The specificity to the E3 protein was confirmed by Western blotting and immunofluorescence of cells infected with either wild-type vaccinia virus or a mutant virus with the E3L gene deleted. Antibody reactivity with E3 was also seen in cells transfected with a plasmid expressing the E3 protein. A panel of mutated vaccinia viruses with truncations in the E3L gene revealed that while MAb 3015B2 reacted with E3 lacking the C-terminal 7 amino acids, it lost reactivity with a mutant E3 lacking the C-terminal 26 amino acids. This indicates that the antigenic site recognized by 3015B2 is on the C-terminus, somewhere between amino acids 164 through 183. The antibody also recognizes the E3 protein encoded by other orthopoxviruses. This antibody will be useful for further investigations of the E3 protein as well as a useful reagent to indicate vaccinia virus early protein expression.