Activity-based selection of HIV-1 reverse transcriptase variants with decreased polymerization fidelity

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Human Immunodeficiency Virus type 1 (HIV-1) reverse transcriptase (RT) is crucial for viral replication, exhibiting high error rates.
• This error-prone nature drives rapid viral evolution, enabling immune evasion and antiviral resistance.
• However, excessive mutations could potentially lead to viral extinction.

Purpose of the Study:

• To identify HIV-1 RT variants with enhanced error rates using genetic selection.
• To investigate the relationship between increased mutation rates and viral replication fidelity.
• To understand the molecular mechanisms underlying altered HIV-1 RT fidelity.

Main Methods:

• A genetic selection scheme was employed to screen for HIV-1 RT mutator variants.
• Candidate variants were purified and characterized through in vitro enzymatic assays.
• Site-directed mutagenesis was used to pinpoint specific amino acid residues responsible for altered enzyme activity.

Main Results:

• Sixteen potential mutator candidates of HIV-1 RT were identified.
• One variant enzyme demonstrated a significantly increased mutation frequency compared to the wild-type enzyme.
• A single amino acid substitution at residue R448, located in the RNase H domain, was implicated in the heightened error rate.

Conclusions:

• The R448 residue plays a critical role in maintaining the fidelity of HIV-1 RT.
• Perturbation of the R448 residue affects template RNA interaction, impacting polymerase activity and fidelity.
• Understanding these fidelity mechanisms could offer insights into viral evolution and novel therapeutic strategies against HIV-1.