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

摘要

Human immunodeficiency virus type 1 (HIV-1) is the cause of an estimated 1.9 millionuddeaths in the world annually. The HI virus has three enzymes, notably; reverseudtranscriptase, integrase and protease, which are crucial for its maturation and continuedudinfection. HIV relies on the catalytic efficacy of the protease enzyme for cleavingudprecursor polyproteins to yield structural and functional proteins. Inhibition of the viraludprotease proffers major therapeutic benefits in the battle against HIV. However, the useudof protease inhibitors in HIV regimens is limited by the emergence of drug resistantudmutations in the protease coding region. Drug resistant mutations are characterized byudamino acid substitutions, deletions, and/or insertions in the viral sequence. Thus, thisudstudy set out to assess structural and functional characteristics of an HIV-1 South Africanudsubtype C (C-SA) protease with a single amino acid substituted and another inserted atudcodon 36, i.e., I36TT, with respect to the wild-type HIV-1 C-SA protease. An I36TTudprotease was generated with its background polymorphisms (P39S, D60E, and Q61E),udover-expressed, and purified. Secondary and tertiary structural properties of the wild-typeudand the variant protease were evaluated using far-UV circular dichroism and fluorescenceudspectroscopy. Both proteases exhibited typical secondary structural features of audpredominantly β-sheeted protein, indicated by circular dichroism spectra with minima atud216 nm. Using intrinsic tryptophan as a probe, the tertiary structures of both proteasesudrevealed that the local structural environments of both proteases had not been perturbed.udThis was indicated by fluorescence emission intensity peak at 355 nm. Proteolyticudefficiency of the protease enzymes was evaluated following hydrolysis of a syntheticudchromogenic HIV substrate mimicking the conserved protease cleavage site in the gagpoludpolyprotein precursor. A comparison of the kinetic properties of the enzymesudindicated that the I36TT variant protease has a slightly (7%) enhanced catalytic activityudrelative to the wild-type HIV-1 C-SA protease. Enzymatic parameters of the twoudproteases in the presence of various protease-inhibitors showed that both proteases’udcatalytic activity is highly affected by saquinavir with IC50 values of 7.6 nM and 6.3 nMudfor the wild-type and the variant, respectively. The variant protease enzyme, compared toudthe wild-type, appears to have acquired resistance towards indinavir with IC50 value ofud16.2 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 itsudenzymatic characteristic as compared to the wild-type protease (IC50 value of 19.1 nM).udThe wild-type and variant protease enzymes showed similar susceptibility (IC50 value ofud17.3 and 16.8 nM for wild-type and variant, respectively) to nelfinavir. Thermodynamicudanalysis of the protease enzymes indicated that the I36TT mutation does not affect theudbinding energetics of acetyl pepstatin to the protease. Thus, vitality studies suggest thatudthe I36TT substitution/insertion mutation and background polymorphisms wereudincorporated in the HIV-1 C-SA protease enzyme to improve viral replication rate.