Structural and functional effects of an I36TT insertion in the South African HIV-1 subtype C protease
Date
2010-09-07
Authors
Mpye, Keleabetswe Lerato
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Abstract
Human immunodeficiency virus type 1 (HIV-1) is the cause of an estimated 1.9 million
deaths in the world annually. The HI virus has three enzymes, notably; reverse
transcriptase, integrase and protease, which are crucial for its maturation and continued
infection. HIV relies on the catalytic efficacy of the protease enzyme for cleaving
precursor polyproteins to yield structural and functional proteins. Inhibition of the viral
protease proffers major therapeutic benefits in the battle against HIV. However, the use
of protease inhibitors in HIV regimens is limited by the emergence of drug resistant
mutations in the protease coding region. Drug resistant mutations are characterized by
amino acid substitutions, deletions, and/or insertions in the viral sequence. Thus, this
study set out to assess structural and functional characteristics of an HIV-1 South African
subtype C (C-SA) protease with a single amino acid substituted and another inserted at
codon 36, i.e., I36TT, with respect to the wild-type HIV-1 C-SA protease. An I36TT
protease was generated with its background polymorphisms (P39S, D60E, and Q61E),
over-expressed, and purified. Secondary and tertiary structural properties of the wild-type
and the variant protease were evaluated using far-UV circular dichroism and fluorescence
spectroscopy. Both proteases exhibited typical secondary structural features of a
predominantly β-sheeted protein, indicated by circular dichroism spectra with minima at
216 nm. Using intrinsic tryptophan as a probe, the tertiary structures of both proteases
revealed that the local structural environments of both proteases had not been perturbed.
This was indicated by fluorescence emission intensity peak at 355 nm. Proteolytic
efficiency of the protease enzymes was evaluated following hydrolysis of a synthetic
chromogenic HIV substrate mimicking the conserved protease cleavage site in the gagpol
polyprotein precursor. A comparison of the kinetic properties of the enzymes
indicated that the I36TT variant protease has a slightly (7%) enhanced catalytic activity
relative to the wild-type HIV-1 C-SA protease. Enzymatic parameters of the two
proteases in the presence of various protease-inhibitors showed that both proteases’
catalytic activity is highly affected by saquinavir with IC50 values of 7.6 nM and 6.3 nM
for the wild-type and the variant, respectively. The variant protease enzyme, compared to
the wild-type, appears to have acquired resistance towards indinavir with IC50 value of
16.2 nM and 9.5 nM for the variant and wild-type, respectively. In the presence of ritonavir, the variant protease (IC50 value of 36.4 nM) proved to have retained most of its
enzymatic characteristic as compared to the wild-type protease (IC50 value of 19.1 nM).
The wild-type and variant protease enzymes showed similar susceptibility (IC50 value of
17.3 and 16.8 nM for wild-type and variant, respectively) to nelfinavir. Thermodynamic
analysis of the protease enzymes indicated that the I36TT mutation does not affect the
binding energetics of acetyl pepstatin to the protease. Thus, vitality studies suggest that
the I36TT substitution/insertion mutation and background polymorphisms were
incorporated in the HIV-1 C-SA protease enzyme to improve viral replication rate.