Analysis of the role of the amino-terminal peptide of vaccinia virus structural protein precursors during proteolytic processing.
Several VV structural proteins are produced by the removal of amino-terminal peptides from their cognate precursors. In the experiments reported here, directed genetic approaches were used to investigate the possible role of these terminal peptides in protein processing. As a model system, the FLAG epitope-tagged P25K precursor was used to prepare constructs in which the 31-amino-acid P25K N-terminal peptide was removed or replaced by heterologous sequences, while the -A-G*-A- cleavage motif was retained. Only a trace amount of the leaderless P25KF(delta 31) polypeptide was found within the mature virions, implying that proteolytic processing is necessary for the incorporation of the 25K product into mature virions. In trans-processing assays, significant levels of the 25K product were generated from wild-type P25KF and P4b:25KF, which consists of the 61-amino-acid P4b terminal peptide, and from P4b:25KF with 15, 30, or 44 residues of the amino terminus deleted. In contrast, only a small amount of 25K was produced from the TK:25KF, which contains the amino-terminal 30 residues of VV thymidine kinase, a protein which is not cleaved under normal circumstances. Furthermore, it has been hypothesized that a hydrophobic residue is required at position P4 relative to the -A-G*-A- motif for the cleavage to take place. An intermediate level of the 25K product was detected from the TK:25KF(Q29V) mutant which has the glutamine residue at P4 replaced with a valine residue, suggesting that the hydrophobic P4 residue and additional substrate determinants in the N-terminal peptide region are required for the proteolytic processing reaction to occur normally. Taken together, these data suggest that the amino-terminal peptides of the VV core proteins are to some extent interchangeable and that the residues proximal to the AGA site are of most importance.
Lee
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Department of Microbiology, Oregon State University, Corvallis 97331-3804.
Hruby
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D000595:Amino Acid Sequence; D002460:Cell Line; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D021382:Protein Sorting Signals / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000502:physiology; D014616:Vaccinia virus / Q000502:physiology*; D015678:Viral Structural Proteins / Q000737:chemistry / Q000378:metabolism*; D014771:Virion; D014779:Virus Replication / Q000502:physiology
D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins