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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.

All proteinases of avian and mammalian retroviruses belong to the family of aspartic proteinases, are of similar size and of homologous primary structure; they all act catalytically in the form of highly symmetric molecular dimers.1 Detailed studies of retroviral proteinases were carried out on two almost identical proteinases of MAV2,3 and RS V4 (representing the group of avian retroviruses) and on the HIV proteinase.5,6 The knowledge of the 3D structure,2,4,5 catalytic activity and substrate specifity3,6 of the MAV and the HIV proteinase has changed the notion of their general similarity since several features that distinguish each proteinase from the other were revealed. The HIV-1 proteinase has a considerably higher activity3,6 which reflects the different conditions of the expression and action of this enzyme in vivo: 7 The “coding strategy” of MAV allows the expression of the proteinase from the first (gag) open reading frame and provides for the high (i.e. stoichiometrical) level of the relatively “weak” enzyme whereas the smaller amount of the more active HIV enzyme is a result of infrequent translational frameshift events that occur in the overlapping region of the gag and pol reading frames.8 The substrate specificities of retroviral proteinases seem complex and the requirement for a side chain in an individual subsite of a substrate is an outcome of the combination of residues occupying other closely located subsites.3 The two proteinases (MAV and HIV) show rather promiscuous substrate specificity, nevertheless several differences can be traced. We made an attempt to use protein engineering of the MAV proteinase to tackle directly problems of structural basis of these differences and, vice versa, to make more precise conclusions on the functional importance of the individual elements of its three dimensional structure. This article describes mutation of the MAV proteinase which resulted not only in an alteration of its substrate specificity but also in an increase of its enzymic activity — a rare case in protein engineering.