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Mechanistic understanding of an altered fidelity simian immunodeficiency virus reverse transcriptase mutation, V148I,

Tracy L Diamond1, George Souroullas, Kellie K Weiss

  • 1Department of Microbiology and Immunology, University of Rochester, New York, 14642, USA.

The Journal of Biological Chemistry
|May 13, 2003
PubMed

Insights

A specific mutation, V148I, in the simian immunodeficiency virus reverse transcriptase (RT) significantly increases its fidelity. This enhanced fidelity results from reduced binding to deoxynucleotide triphosphates (dNTPs), impacting viral replication.

Area of Science:

  • Virology
  • Molecular Biology
  • Biochemistry

Background:

  • The reverse transcriptase (RT) of SIVMNE 170 exhibits higher fidelity than the parental SIVMNE CL8 strain.
  • Previous research identified residue 151 as crucial for RT fidelity and deoxynucleotide triphosphate (dNTP) binding.

Purpose of the Study:

  • To elucidate the mechanistic basis for the increased fidelity of the SIVMNE 170 RT variant.
  • To identify specific mutations responsible for the enhanced fidelity and understand their impact on RT function.

Main Methods:

  • Site-directed mutagenesis to isolate the effect of individual mutations.
  • Pre-steady state kinetic analysis to compare dNTP binding affinity (Kd) and catalysis (kpol) between wild-type and mutant RTs.
  • Analysis of amino acid residue 148's role in RT fidelity.

Main Results:

  • The V148I mutation was identified as the sole determinant of the increased fidelity in the 170 RT variant.
  • The V148I mutation significantly decreased the binding affinity (Kd) of RT to both correct and incorrect dNTPs, without affecting catalytic rate (kpol).
  • Valine at position 148 is essential for RT infidelity, while its mutation to isoleucine enhances fidelity.

Conclusions:

  • The V148I mutation in SIV RT enhances fidelity by reducing dNTP binding affinity, similar to effects observed with Q151N mutations.
  • Alterations at residue 148 critically influence RT-dNTP interactions, potentially affecting viral replication and fitness.
  • Understanding these molecular mechanisms provides insights into viral evolution and adaptation.

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