The
Ad2 proteinase, which is thought to be encoded by a 23-kDa open reading frame located at the end of the L3 family of late mRNAs, is expressed poorly even late after
infection. To obtain sufficient
proteinase for biochemical characterization,
a DNA fragment containing the 23-kDa open reading frame was cloned into plasmids that permit efficient expression in Escherichia coli. Polyclonal antiserum specific for the
Ad2 proteinase was produced by immunizing rabbits with a fusion
protein that included the entire
proteinase open reading frame, and this antiserum was used to show that the product of the 23-kDa reading frame is assembled into virions. Bacterial products corresponding to the complete 204
amino acid proteinase reading frame, to a 9
amino acid proteinase deletion, and to a
proteinase fusion
protein of 227
amino acids were used to determine the size of the
proteinase polypeptide in Ad2 virions and in infected HeLa cell extracts. A single
proteinase polypeptide that migrated during SDS-
polyacrylamide gel electrophoresis with the 204
amino acid recombinant
proteinase was detected in wild-type and H2ts1 virions, and in infected
cell extracts. Immunoblot titrations showed that a wild-type Ad2 virus particle contains about 10
proteinase polypeptides; an H2ts1 virion has approximately fivefold less
proteinase. In virions, the
proteinase was associated primarily with the virus core. The 204
amino acid proteinase produced in E. coli permitted cleavage of the
major core protein precursor, P-VII, to mature, authentic VII, but the
proteinase deletion lacking 9
amino acids from near the amino-terminus was inactive. These results are inconsistent with autocatalytic processing of the
Ad2 proteinase as was reported by Chatterjee and Flint (1987, Proc. Natl. Acad. Sci. USA 84, 714-718).