Unequal human immunodeficiency virus type 1 reverse transcriptase error rates with RNA and DNA templates

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Sequence variation in human immunodeficiency virus type 1 (HIV-1) arises partly from reverse transcriptase (RT) inaccuracies.
• HIV-1 RT is known to be error-prone with DNA templates in vitro, but its fidelity during RNA-dependent DNA synthesis in the viral life cycle is less understood.

Purpose of the Study:

• To compare the fidelity of HIV-1 reverse transcriptase using RNA and DNA templates of identical sequences.
• To investigate the impact of template type on specific error types, such as substitutions and frameshifts.

Main Methods:

• Development of a system to assess the fidelity of both transcription and reverse transcription.
• Comparative analysis of DNA synthesis fidelity by HIV-1 RT using RNA versus DNA templates.
• Sequence analysis of generated mutants to identify error patterns.

Main Results:

• HIV-1 reverse transcriptase exhibited several-fold higher fidelity with RNA templates compared to DNA templates.
• Specific errors, including substitutions and single-nucleotide deletions at homopolymeric regions, were over 10-fold less frequent with RNA templates.
• Initial nucleotide incorporation during RNA-templated synthesis showed increased error rates, possibly due to early enzyme-substrate interaction issues.

Conclusions:

• The fidelity difference between RNA and DNA templates suggests that errors during minus-strand and plus-strand DNA synthesis may contribute unequally to HIV-1 mutation rates.
• Template-primer slippage appears less frequent with RNA templates, indicating differences in intermediate formation or utilization.
• The study also provides error rate estimates for T7 RNA polymerase transcription.