Stepwise mechanism of HIV reverse transcriptase: primer function of phosphorothioate oligodeoxynucleotide

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Human Immunodeficiency Virus (HIV) reverse transcriptase is a key enzyme for viral replication.
• Understanding enzyme-primer interactions is crucial for developing antiviral therapies.
• Previous studies established an ordered reaction pathway for DNA synthesis by HIV reverse transcriptase.

Purpose of the Study:

• To investigate primer recognition by purified HIV reverse transcriptase.
• To characterize the inhibitory effects of oligodeoxynucleotides on DNA synthesis.
• To determine kinetic parameters of enzyme-primer interactions.

Main Methods:

• Enzyme kinetics studies using various template-primers.
• Competitive inhibition assays with d(C)28 and phosphorothioate oligodeoxynucleotide Sd(C)28.
• Filter binding assays to confirm enzyme-oligonucleotide binding affinity.
• Substrate kinetic studies to determine Km values.

Main Results:

• d(C)28 acts as a linear competitive inhibitor, binding to the free enzyme.
• Sd(C)28 demonstrates significantly higher affinity (lower Ki ≈ 2.8 nM) than d(C)28, indicating potent inhibition.
• Kinetic analysis revealed a Km of 24 nM for Sd(C)28 as a primer, with association and dissociation rate constants determined.

Conclusions:

• Phosphorothioate oligodeoxynucleotides, specifically Sd(C)28, are high-affinity binders and potent competitive inhibitors of HIV reverse transcriptase.
• The kinetic data provides insights into the real-time dynamics of enzyme-primer association and dissociation.
• These findings contribute to understanding HIV reverse transcriptase mechanisms and potential therapeutic targets.