Martin Švec1,2, Kvido Stříšovský1 and Jan Konvalinka1
1) Institute of Organic Chemistry and Biochemistry, Flemingovo nam. 2, Prague 6, 166 10, Czech Republic
2) Centre for Complex Molecular Systems and Biomolecules, Flemingovo nam. 2, Prague 6, 166 10, Czech Republic
Murine intracisternal type A particles (intracisternal A-particles, IAPs) are endogenous retroviruses encoded by many proviral elements within the mouse genome. They share sequence homology with the B-type mouse mammary tumor virus (MMTV), the D-type simian retroviruses (SRV) and C-type avian sarcoma virus. IAPs genetic elements have been shown to transpose within the genome of retrovirus-producing cells.
The IAP particles assemble and bud at the membranes of the endoplasmic reticulum (ER) where they accumulate as immature particles consisting exclusively of uncleaved polyproteins. They do not leave cell and horizontal transmission via free particles has not been achieved. Recent evidence has indicated that the lack of proteolytic processing is not due to a defective viral proteinase but rather is caused by the site of particle formation and can be rescued by an artificial redirection of the polyprotein to the plasmatic membrane.
The recombinant proteinase of murine intracisternal A-type particle 14 (MIA14 PR) undergoes N- and C- terminal autoprocessing at defined sites and is sequentially and functionally related to the B- and D-type retrovirus proteinases. An unusual feature of these proteinases as opposed to the C-type retrovirus proteinases is a 50 amino acid C-terminal extension of unknown function. In this study, we aim to analyse the possible role of the C-terminal extension of the proteinase in regulation of polyprotein processing.
We have cloned, expressed in E. coli and purified to homogeneity both the full-length MIA14 PR and its C-terminally truncated form. Both enzymes are active and have been used for in vitro kinetic studies using peptide substrates and inhibitors. The C-terminal extension of MIA14 PR has been cloned, expressed in E. coli, purified and its effect on catalytic activity of both MIA14 PR constructs evaluated.