Studies on the substrate specificity of the proteinase of equine infectious anemia virus using oligopeptide substrates

Abstract

The proteinase of the equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus (HIV), was purified from concentrated virus. The specificity of the enzyme was characterized using oligopeptides representing naturally occurring cleavage sites in the Gag and Gag-Pol polyproteins. The length of the substrate binding pocket was found to be 1-2 residues longer than that of HIV proteinases. Although the EIAV and HIV proteinases cleaved most of the peptides at the same bond, some were hydrolyzed by only the EIAV enzyme. Oligopeptides representing cleavage sites in the nucleocapsid protein were also found to be substrates of the EIAV proteinase. However, these peptides were not hydrolyzed by the HIV proteinases. While peptides representing the corresponding sequences in the first cysteine arrays of the nucleocapsid proteins of HIV-1 and HIV-2 were substrates of the proteinases, peptides representing the homologous sequences in the second Cys arrays were resistant against the proteolytic attack. A three-dimensional model of the EIAV proteinase built on the basis of homology with HIV-1 proteinase was used to interpret the differences. In addition to the oligopeptides representing cleavage sites in the Gag and Gag-Pol polyproteins, the EIAV proteinase was also able to cleave an oligopeptide mimicking a cleavage site in the transmembrane protein. Our results suggest that the specificity of lentiviral proteinases share common characteristics, although substantial differences may exist in hydrolysis of some peptides.

Expression, Characterisation and Mutagenesis of the Aspartic Proteinase from Equine Infectious Anaemia Virus

“…The larger amounts of EIAV proteinase purified from E. coli in the present study not only permitted the demonstration that the activity of the recombinant enzyme was directly comparable to that of its natural, viral, counterpart [3] but also facilitated a much more extensive characterisation of the enzyme than was possible hitherto. However, the size, Nterminal sequence and crystal structure determined for the recombinant EIAV proteinases confirm the identification of the cleavage junctions at the N-terminus of proteinase and at its Cterminal junction with reverse transcriptase [3]. Furthermore, in attempting to predict the cleavage junctions in the EIAV polyprotein using peptide substrates as mimics, Tozser et a1.…”

“…1 ) with that predicted from the oligonucleotide sequence of the cDNA ; furthermore. the N-terminus of the recombinant proteinase is exactly coincident with the predicted cleavage junction in EIAV pol for the start of proteinase [3]. In some cases, the purified protein was Table 1.…”

“…The isolation of small amounts of EIAV proteinase from the virus itself has been described previously [3] ; these investigators demonstrated the ability of the naturally occurring viral proteinase to hydrolyse synthetic peptides mimicking the (putative) cleavage junctions of the EIAV polyprotein, with varying degrees of efficiency. The larger amounts of EIAV proteinase purified from E. coli in the present study not only permitted the demonstration that the activity of the recombinant enzyme was directly comparable to that of its natural, viral, counterpart [3] but also facilitated a much more extensive characterisation of the enzyme than was possible hitherto.…”

“…Furthermore, in attempting to predict the cleavage junctions in the EIAV polyprotein using peptide substrates as mimics, Tozser et a1. [3] suggested that (a) some of the sites might be displaced relative to those in HIV and (b) that the cleavage junction between the p51 subunit of EIAV reverse-transcriptase and the RNase H domain was located approximately 15 residues further downstream from its counterpart in the HIV-1 polyprotein EIAV:…”

“…The Tyr-Pro bond between the matrix and capsid proteins of EIAV (and HIV) gag is an example of the former, whereas the C-terminus of the capsid protein is connected to the next protein in the gag precursor by a Leu-Leu bond [3], illustrative of the hydrophobic-hydrophobic type of cleavage junction. Peptides mimicking the sequence at each of these two representative cleavage junctions in EIAV gag were synthesised and kinetic parameters for their hydrolysis by wild-type EIAV proteinase were determined.…”