Abstract
We have cloned and expressed the 3' region of the Mason-Pfizer monkey virus pro gene in Escherichia coli. The recombinant 26-kDa precursor undergoes rapid self-processing both in E. coli and in vitro at the NH2 terminus, yielding a proteolytically active 17-kDa protein, p17. This initial cleavage is followed in vitro by a much slower self-processing that leads to emergence of proteolytically active p12 and a COOH-terminal cleavage product p5. We have found the NH2-terminal processing site of both the p17 and p12 to be identical and similar to the amino terminus of the mouse mammary tumor virus proteinase. We have also identified the COOH-terminal processing site of the p12 form. Using purified recombinant proteins and synthetic oligopeptide substrates based on naturally occurring retroviral processing sites, we have determined the enzymatic activity and specificity of the Mason-Pfizer monkey virus proteinase to be more closely related to that of myeloblastosis-associated virus proteinase rather than that of the Human immunodeficiency virus type 1 proteinase. Inhibition studies using peptide inhibitors support these results.
Highlights
We have cloned and expressed the 3' region of the Mason-Pfizer monkey virus pro gene in Escherichia coli
Cloning-Construction of the bacterial expression vector pGOT7Q was carried out in consideration of the unknown molecular weight and termini ofthe M-PMV PR and under the presumption that M-PMV PR would be toxic for E. coli, as has been demonstrated for other retroviral proteinases [9, 12]
M-PMV is a prototypic type D retrovirus that, in a manner similar to the B-type MMTV, assembles immature ICAPs in the cell cytoplasm, and transports these particles to the cellular membrane, where they undergo proteolytic maturation during release from the cell. This arrangement requires a very tightly regulated activation of the virally programmed proteolytic eventrs), does M-PMV PR cleave gag gene-related precursors [1], its activity is necessary for the removal of a carboxyl-terminal segment from the transmembrane glycoprotein, gp22, resulting in a virus-associated gp20 [1, 4]
Summary
Ferring-Leciva, a.s., Prague Polypeptide Institute, Komoranska 955, 14310 Prague, Czech Republic. During or shortly after particle release from the cell, the viral PR, as part of Gag-Pro and Gag-Pro-Pol precursors, is activated and cleaves the precursor polypeptides into their constituent proteins. In M-PMV PR-deficient virions, do the Gag-containing polypeptides remain unprocessed, the transmembrane glycoprotein, which is initially synthesized as a cellassociated gp, is not cleaved within the cytoplasmic domain to generate the gp found in wild-type virions (I, 4) While these studies provided evidence that M-PMV PR belongs to the aspartyl proteinase family, they provided little information on the biochemical and structural properties of this enzyme. The purified proteins have been used to determine the substrate specificity of the enzyme and its relatedness to other retroviral enzymes
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