Abstract
The ordered, sequential cleavages of the Gag-Pol polyprotein by human immunodeficiency virus (HIV) protease present the virus with severe limitations on viable mutations of the enzyme. An extension of the method of Kuchel et al. (Kuchel, P. W., Nichol, L. W., and Jeffrey, P. D. (1974) J. Theor. Biol. 48, 39-49) for the analysis of consecutive enzyme reactions leads to a simple description of the catalytic efficiency of mutant and wild type HIV protease in the presence or absence of inhibitors. The overall catalytic efficiency of a mutant HIV protease relative to the wild type enzyme is given by the product of the ratios of their respective efficiencies for the 8 obligatory cleavages. Under no conditions is HIV viable when the geometric mean efficiency of a mutant HIV protease is less than 61% of the wild type activity for each cleavage. The lower catalytic efficiencies of the mutant enzymes coupled with the exponential dependence on 1/(1 + [I]/Ki) more than offset the inhibitor resistance acquired by HIV protease. The conclusion of this analysis is that inhibitor-resistant mutant HIV proteases are very unlikely to contribute to viral viability in vivo. The results strongly suggest that future protease inhibitor clinical trials should measure the infectivity of the virions in blood plasma instead of relying on viral RNA levels.
Highlights
A number of investigators have attempted to quantify the effects of mutations on the kinetics of human immunodeficiency virus (HIV) protease, both in the presence and absence of inhibitors [3, 6, 7, 21,22,23]
Gulnik et al [22] point out that, “while inhibition constants should not depend on the substrate, kcat/Km ratios do . . . future studies should focus on a panel of substrates that represent all the natural HIV cleavage sites in the Gag-Pol polyproteins.”
At least eight obligatory cleavages of the Gag-Pol polyprotein by HIV protease have been identified for viable maturation of viral particles [24], and there is considerable evidence that the order of the eight cleavages is not random
Summary
A number of investigators have attempted to quantify the effects of mutations on the kinetics of HIV protease, both in the presence and absence of inhibitors [3, 6, 7, 21,22,23]. At least eight obligatory cleavages of the Gag-Pol polyprotein by HIV protease have been identified for viable maturation of viral particles [24], and there is considerable evidence that the order of the eight cleavages is not random. In vitro studies clearly show time-dependent, sequential formation and disappearance of the various intermediates during processing of the Gag-Pol polyprotein by HIV protease [25,26,27,28]. While HIV protease can cleave its viral proteins in a variety of ways, depending on the conditions, it is proposed that only a specific, ordered sequence of Gag-Pol polyprotein processing leads to infectious virions. The ordered processing sequence is kinetically equivalent to a series of
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