Through the use of transient expression assays and directed genetics, the vaccinia virus (VV) I7L gene product has been implicated as the major maturational proteinase required for viral core protein cleavage to occur during virion assembly. To confirm this hypothesis and to enable a biochemical examination of the I7L cysteine proteinase, an in vitro cleavage assay was developed. Using extracts of VV infected cells as the source of enzyme, reaction conditions were developed which allowed accurate and efficient cleavage of exogenously added core protein precursors (P4a, P4b and P25K). The cleavage reaction proceeded in a time-dependent manner and was optimal when incubated at 25 degrees C. I7L-mediated cleavage was not affected by selected inhibitors of metalloproteinases, aspartic acid proteinases or serine proteinases (EDTA, pepstatin, and PMSF, respectively), but was sensitive to several general cysteine proteinase inhibitors (E-64, EST, Iodoacetic acid, and NEM) as well as the I7L active site inhibitor TTP-6171 [C. Byrd et al., J. Virol. 78:12147-12156 (2004)]. Finally, in antibody pull down experiments, it could be demonstrated that monospecific alphaI7L serum depleted the enzyme activity whereas control sera including alphaG1L, directed against the VV metalloproteinase, did not. Taken together, these data provide biochemical evidence that I7L is a cysteine proteinase which is directly involved in VV core protein cleavage. Furthermore, establishment of this I7L-mediated in vitro cleavage assay should enable future studies into the enzymology and co-factor requirements of the proteolysis reaction, and facilitate antiviral drug development against this essential target.