The K65R mutation confers increased DNA polymerase processivity to HIV-1 reverse transcriptase

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• The K65R mutation in HIV-1 reverse transcriptase (RT) confers cross-resistance to multiple nucleoside reverse transcriptase inhibitors (NRTIs).
• Understanding the biochemical properties of mutant RT is crucial for developing effective HIV-1 therapies.

Purpose of the Study:

• To investigate the impact of the K65R mutation on the processivity and kinetics of HIV-1 reverse transcriptase during DNA synthesis.
• To elucidate the molecular mechanisms underlying viral resistance and replication in the presence of the K65R mutation.

Main Methods:

• In vitro DNA synthesis assays using wild-type (wt) and K65R mutant HIV-1 RT.
• Analysis of nucleotide incorporation, processivity, and template/primer (T/P) dissociation rates.
• Comparison of enzyme kinetics under single processive cycle and continuous DNA synthesis conditions.

Main Results:

• K65R RT exhibited significantly higher processivity compared to wt RT, with 20-50% greater nucleotide incorporation under single processive cycle conditions.
• The rate of full-length product synthesis by K65R RT was approximately 2-fold higher than wt RT under single processive cycle conditions.
• A decreased rate of T/P dissociation was observed for K65R RT, correlating with its enhanced processivity.

Conclusions:

• The increased processivity of K65R RT may serve as a compensatory mechanism for reduced dNTP substrate affinity.
• This enhanced processivity could contribute to maintaining viral replication kinetics in the presence of resistance mutations.
• Findings provide insights into the complex interplay between HIV-1 RT mutations, drug resistance, and viral fitness.