Structural and functional effects of an I36TT insertion in the South African HIV-1 subtype C protease

Simple item page
dc.contributor.authorMpye, Keleabetswe Lerato
dc.date.accessioned2010-09-07T05:35:51Z
dc.date.available2010-09-07T05:35:51Z
dc.date.issued2010-09-07
dc.description.abstractHuman 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.en_US
dc.identifier.urihttp://hdl.handle.net/10539/8617
dc.language.isoenen_US
dc.titleStructural and functional effects of an I36TT insertion in the South African HIV-1 subtype C proteaseen_US
dc.typeThesisen_US
Files
Original bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
Corrected MSc dissertation - Mpye K.L.pdf
Size:
4.12 MB
Format:
Adobe Portable Document Format
Download
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
license.txt
Size:
1.71 KB
Format:
Item-specific license agreed upon to submission
Description:
Download
Collections
ETD Collection