Journal of Biotechnology & Biomaterials
Open Access

Abstract

The unique properties of human immunodeficiency virus type 1 reverse trancriptase (HIV-1 RT) include its high propensity for misinsertion and misincorporation of deoxyribonucleotide triphosphates (dNTP) in the growing chain’s 3’ terminus. It was envisaged that the interaction of the side chain of K154 in HIV-1RT with the penultimate nucleotide of template may be crucial in determination of fidelity of proviral cDNA synthesis. This hypothesis was tested by steady-state kinetic studies using wild-type HIV-1 RT and five K154 mutants. These mutants contained replacement of positively charged side chain of Lysine with two amino acids’ hydrophobic and two amino acids’ negatively charged side chains. In one of the mutants, the positive charge of Lysine was retained but the side chain was extended by one carbon atom while replacing it with Arginine. The results indicated that the mutants with negatively charged side chains displayed significant decrease in enzyme activity whereas other mutants exhibited enzyme activities almost comparable to the wild type. It was observed that excepting the mutants with negatively charged side chains which displayed higher fidelity than wild type, all other mutants showed enhanced levels of misinsertion and mispair extension; K154R being the more prominent. All mutants when tested for their response to an approved antiHIV-RT agent i.e., 3TC, reflected significant resistance to this nucleotide analog when compared to wild type enzyme. The mechanisms of misinsertion, mispair extension as well as drug resistance of these mutants would be explained in the light of three dimensional crystal structures of HIV-1RT.

Biography

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