Human immunodeficiency virus type 1 proteinase : regulation of function and catalytic activity
Author: Goobar-Larsson, Laura
Date: 1996-03-29
Location: Bakteriologens
Time: 9.30
Abstract
HIV-l proteinase activity is a prerequisite for viral replication, and
one of the main targets for anti-HlV therapy. Although potent proteinase
inhibitors are currently being approved for treatment of AIDS patients,
these drugs are unlikely to provide treatment which can give patients a
life of normal quality and duration. The development of improved drugs
against HIV/AIDS requires further understanding of the function and
activity of their targets. The work of this thesis has focused on the
identification and characterisation of different factors that regulate
HIV-l proteinase function and activity.
Methods for expression and purification of HIV-l proteinase from bacteria
were developed, and proteinase activity in vitro was measured using
synthetic peptides representing sequences of the naturally occurring
cleavage sites.
HIV-l and AMV proteinases were found to inhibit microtubule assembly in
vitro, and this inhibition was associated with cleavage of microtubule-
associated proteins 1 and 2. These results showed that the substrate
specificity of HIV-l proteinase in vitro was not restricted to viral
proteins, and indicated a potential cytotoxicity of the proteinase during
viral infection.
The substrate specificity of HIV-l proteinase is not only determined by
the amino acid sequence of the cleavage site and by its secondary
structure, but also by its accessibility. It is the tertiary or
quaternary structure of the substrate protein which will ultimately
determine whether a site will be cleaved or not. Such a structural
determinant regulating the limited cleavage of p66 RT to p5 1 RT was
identified, and consisted of a salt bridge between D488 and L465 located
just above the proteinase cleavage site.
HIV-l RT was found to enhance proteinase activity in a dose-dependent
manner both in vitro and in cell culture. The degree of enhancement was
substrate-dependent, with the lowest effect on the proteinase
autocleavage site and the site between the proteinase and RT, and with
the highest effect on the cleavage site between RT and the integrase, and
between p5 1 RT and RNase H. RT enhancement of proteinase activity was
independent of pH and ionic strength, and was not due to an increase in
proteinase dimerization. RT could enhance the activity of a tethered
proteinase dimer and of the wild type proteinase to the same degree, by
increasing their kcat's and decreasing their KM'S.
Cross-linking experiments of RT and proteinase showed that the proteinase
cross-linked mainly to the monomer forms of RT. Studies of deletion
mutants indicated that sequences in the polymerase and RNase H domain of
RT are needed for maximal effect on proteinase activity. Three putative
interacting sites in the RNase H domain of RT were identified using
overlapping peptides coding for the entire RT sequence. The most active
peptide corresponded to the region of RNase H that had to be unravelled
in order for processing of RT to occur. These results suggest that in
vivo, RT may regulate its own cleavage from the polyprotein precursor.
At low ionic strength, and after equilibration at 37¡, proteinase
activity in vitro was found to deviate from normal Michaelis-Menten
kinetics even though activity was stable under the conditions used. This
could be overcome by increasing the ionic strength of the solution, or by
inhibiting proteinase dissociation by avoiding preincubation or using a
tethered proteinase dimer. These results indicate that deviation from
normal kinetics is due to changes in the degree of proteinase
dimerization upon addition of substrate, and that at least in vitro,
HIV-l proteinase is activated by its substrate.
Issue date: 1996-03-08
Publication year: 1996
ISBN: 91-628-1911-9
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