Structural determinants of slippage-mediated mutations by human immunodeficiency virus type 1 reverse transcriptase

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Single-base deletions and -1 frameshifting in HIV-1 reverse transcriptase (RT) are critical for viral replication.
• These errors are attributed to template slippage during DNA polymerization.
• The crystal structure of HIV-1 RT indicates a role for Glu(89) in the template cleft, near the slippage site.

Purpose of the Study:

• To investigate the functional role of Glu(89) in HIV-1 RT frameshifting and fidelity.
• To determine how perturbing the interaction of Glu(89) with the template backbone affects frameshift frequencies and nucleotide incorporation.

Main Methods:

• Site-directed mutagenesis was used to create substitutions at Glu(89) in recombinant HIV-1 RT.
• Frameshift frequencies (-1 and +1) were measured for wild-type and mutant RTs.
• Rates of dNTP misincorporation were assessed to evaluate RT fidelity.

Main Results:

• All Glu(89) substitutions significantly reduced -1 frameshifting (2-40-fold).
• Many substitutions enhanced +1 frameshifting (3-47-fold), suggesting Glu(89) influences slippage on both strands.
• Reduced dNTP misincorporation rates correlated with decreased -1 frameshifting, indicating a shared mechanism.

Conclusions:

• Glu(89) plays a significant role in regulating slippage-mediated errors, including -1 frameshifting.
• The fidelity of dNTP incorporation by HIV-1 RT is also influenced by Glu(89).
• These findings highlight Glu(89) as a key determinant of HIV-1 RT error rates and a potential target for antiviral strategies.