Mutations Proximal to Sites of Autoproteolysis and
the α‑Helix That Co-evolve under Drug Pressure Modulate
the Autoprocessing and Vitality of HIV‑1 Protease
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Abstract
N-Terminal self-cleavage
(autoprocessing) of the HIV-1 protease
precursor is crucial for liberating the active dimer. Under drug pressure,
evolving mutations are predicted to modulate autoprocessing, and the
reduced catalytic activity of the mature protease (PR) is likely compensated
by enhanced conformational/dimer stability and reduced susceptibility
to self-degradation (autoproteolysis). One such highly evolved, multidrug
resistant protease, PR20, bears 19 mutations contiguous to sites of
autoproteolysis in retroviral proteases, namely clusters 1–3
comprising residues 30–37, 60–67, and 88–95,
respectively, accounting for 11 of the 19 mutations. By systematically
replacing corresponding clusters in PR with those of PR20, and vice
versa, we assess their influence on the properties mentioned above
and observe no strict correlation. A 10–35-fold decrease in
the cleavage efficiency of peptide substrates by PR20, relative to
PR, is reflected by an only ∼4-fold decrease in the rate of
Gag processing with no change in cleavage order. Importantly, optimal
N-terminal autoprocessing requires all 19 PR20 mutations as evaluated <i>in vitro</i> using the model precursor TFR-PR20 in which PR
is flanked by the transframe region. Substituting PR20 cluster 3 into
TFR-PR (TFR-PR<sup>PR20‑3</sup>) requires the presence of PR20
cluster 1 and/or 2 for autoprocessing. In accordance, substituting
PR clusters 1 and 2 into TFR-PR20 affects the rate of autoprocessing
more drastically (>300-fold) compared to that of TFR-PR<sup>PR20‑3</sup> because of the cumulative effect of eight noncluster mutations present
in TFR-PR20<sup>PR‑12</sup>. Overall, these studies imply that
drug resistance involves a complex synchronized selection of mutations
modulating all of the properties mentioned above governing PR regulation
and function