Human immunodeficiency virus reverse transcriptase. Effect of primer length on template-primer binding.

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) is a crucial enzyme for viral replication.
• Understanding the enzyme's interaction with template-primers is essential for developing antiviral therapies.
• HIV-1 RT exists as a heterodimer (p66-p51) and homodimers (p66 or p51).

Purpose of the Study:

• To investigate the influence of primer length on the binding kinetics of HIV-1 RT to template-primers.
• To elucidate the roles of the p66 and p51 subunits in template-primer binding.
• To determine the contribution of the RNase H domain to enzyme-template-primer interactions.

Main Methods:

• Gel electrophoresis to analyze primer extension patterns.
• Steady-state kinetics and enzyme-template-primer trapping experiments.
• Binding assays using poly(rA).oligo(dT)n and poly(dA).oligo(dT)n with varying oligo(dT)n lengths.

Main Results:

• Binding affinity (Kd) showed a primer length-dependent transition, with a significant increase in affinity for primers of 16-20 nucleotides compared to 10-14 nucleotides.
• The dissociation rate constant was independent of primer length, suggesting the primer length dependence of Kd is due to the association rate.
• The p66 homodimer exhibited primer length-dependent binding similar to the heterodimer, while the p51 homodimer showed primer-length-independent binding.

Conclusions:

• A critical primer length of approximately 14-16 nucleotides exists for high-affinity binding of HIV-1 RT heterodimer and p66 homodimer.
• The RNase H domain of the p66 subunit likely plays a role in the primer length-dependent binding of the heterodimer and p66 homodimer.
• These findings provide insights into the molecular mechanisms of HIV-1 RT and potential targets for drug development.