The proteinase polypeptide of adenovirus serotype 2 virions.
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).
Anderson
C W
CW
Biology Department, Brookhaven National Laboratory, Upton, New York 11973.
D000260:Adenoviruses, Human / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000302:isolation & purification; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013379:Substrate Specificity
D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C091827:adenain