Physical and molecular genetic analysis of the multistep proteolytic maturation pathway utilized by vaccinia virus P4a protein.
Three potential AG*X cleavage sites have previously been identified in vaccinia virus (VV) P4a precursor, namely AG*N, AG*S and AG*T. Utilization of the COOH-proximal AG*T site in P4a leads to release of a 23 kDa product ('23K'). Here we propose that cleavage at the AG*S site alone is responsible for release of 4a, based on peptide mapping and microsequencing which demonstrated that the NH2 terminus of 4a is co-terminal with P4a, thus indicating that the AG*N site is excluded from proteolytic processing. Proteolysis of P4a at AG*S and AG*T to yield 4a and 23K should theoretically also liberate an intervening 9 kDa peptide ('9K'), although efforts to isolate this peptide have been repeatedly unsuccessful. To investigate the fate of this intervening peptide, mutation of the P4a coding sequence at the AG*S or AG*T site, followed by transient expression in VV-infected cells, lead to the synthesis of unique 4a-9K or 9K-23K chimeric protein products. This implies that neither end of the intervening 9K peptide is intrinsically destabilizing, and that its supposed degradation may be suppressed when it remains associated with 4a or 23K.
Whitehead
S S
SS
Department of Microbiology, Oregon State University, Corvallis 97331-3804.
Bersani
N A
NA
Hruby
D E
DE
D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D017421:Sequence Analysis; D014616:Vaccinia virus / Q000378:metabolism*; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C069852:P4a protein, Vaccinia virus; D010446:Peptide Fragments; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins