Abstract
Previous studies have suggested that cleavage of vaccinia virus core protein precursors occurs within the consensus tripeptide motif -A-G decreases X-. As an approach to delineate the sequence and structural features of the precursor polypeptides that are responsible for directing site-specific scission within this element, site-directed mutagenesis procedures were employed in concert with an in vivo trans-processing assay of the P25K: FLAG reporter plasmid. The results obtained suggest that residue occupancy at the P1' site (following the nomenclature of Schechter and Berger (Schechter, I., and Berger, A. (1976) Biochem. Biophys. Res. Commun. 27, 157-162), the positions at the amino- and carboxyl-proximal residues are indicated as P1, P2, etc., and P1', P2', etc., respectively) was extremely permissive, with only a proline substitution blocking cleavage. In contrast, the permissible occupancy of the P1 (serine or alanine) and P2 (cysteine, serine, or asparagine) sites was extremely restricted. Analysis of P1/P2 double mutants supported this conclusion and suggested additional levels of combinatorial stringency. Insertion or deletion of sequences immediately adjacent (amino- or carboxyl-terminal) to the -A-G-X- motif completely abrogated cleavage, suggesting the presence of additional important structural determinants. Mutation of the conserved proline or basic amino acid residues in these regions had no effect on cleavage, whereas it appeared that the presence of a hydrophobic residue in the P4 site was required.
Highlights
From the Center for Gene Research and Biotechnology, Department of Microbiology, Nash Hall 220, Oregon State Uniuersity, Coruallis, Oregon 97331-3804
Sequence has been implicated in the production of a second product, 25K’, from the P25K precursor [2, 9].a consensus -A-G J X-sequence has been proposed in the precursor substrates as the siteof proteinase recognition and hywas extremely restricted
The P25K: processing of W major core proteins is found to be arrested FLAG substrate consists of the authenticP25K precursor plus when virus maturation ipsrevented by the additionof the drug an 8-amino acid epitope (FLAG)tagged t o its carboxyl terminus rifampin [4].Likewise, similar concurrent blockage of core pro- for the detection of transiently expressed P25K derivatives as tein proteolysis and virus maturation were observed in cells opposed to thegenomic P25K derivatives
Summary
(Received for publication, October 12, 1993, and in revised form, December 1, 1993). From the Center for Gene Research and Biotechnology, Department of Microbiology, Nash Hall 220, Oregon State Uniuersity, Coruallis, Oregon 97331-3804. The P25K: processing of W major core proteins is found to be arrested FLAG substrate consists of the authenticP25K precursor plus when virus maturation ipsrevented by the additionof the drug an 8-amino acid epitope (FLAG)tagged t o its carboxyl terminus rifampin [4].Likewise, similar concurrent blockage of core pro- for the detection of transiently expressed P25K derivatives as tein proteolysis and virus maturation were observed in cells opposed to thegenomic P25K derivatives Using this assay, an infected with one subset of temperature-sensitive mutants at additional cleavage product, 25K’, derived from cleavage at the the nonpermissive temperature[5].
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