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
Abstract
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.