Processing, Purification, and Kinetic Characterization of the Gag-Pol Encoded Retroviral Proteinase of Myeloblastosis Associated Virus Expressed in E. Coli
Gag-Pol frameshift translational products of avian retroviruses (e. g. myeloblastosis associated virus, MAV, or Rous sarcoma virus, RSV) contain a putative proteinase species that maps between the NC/PR and PR/RT processing sites. Such Gag-Pol proteinase deffers from the gag-encoded proteinase (Gag PR, 124 amino acids length) by a carboxy-terminal extension of 7 amino acids (numbered 125 to 131), i. e. -IGRATVL (see Refs. 1,2,3, for review). While the main species of the processing proteinase of avian retroviruses, Gag PR (known also as pl5gag) was characterized to considerable details (4, 5, 6, 7), direct description of avian retroviral Gag-Pol proteinases remained very limited (3) and substantial knowledge was missing. We attempted to analyze in detail recombinant gag-pol encoded species of the MAV avian retroviral proteinase (identical to that of RSV, except for a conservative substitution at amino acid residue 84, Ref. 8) using a truncated, E. coli expressed in-frame precursor that retains the natural NC/PR cleavage site and is translationally terminated at the position of the PR/RT site. This work showed that under conditions favourable for processing, an unexpected cleavage occurs between amino acid residues 126 and 127, and that this cleavage bears characteristics of a regular self-processing step. The cleavage product, termed PR(+IleGly), represents an enzyme with catalytic parameters not much different but distinguishable from those of the gag-encoded proteinase.
KeywordsCleavage Product Bovine Leukemia Virus Rous Sarcoma Virus FEBS Letter Amino Acid Length
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