Stabilization of human immunodeficiency virus type 1 reverse transcriptase by site-directed mutagenesis

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a key enzyme in viral replication.
• Current cDNA synthesis relies on Moloney murine leukemia virus RT, but HIV-1 RT variants show higher substrate affinities.
• Enhancing the thermostability of HIV-1 RT could improve its utility in molecular biology applications.

Purpose of the Study:

• To engineer more thermostable variants of HIV-1 group M (HIV-1 M) and HIV-1 group O (HIV-1 O) reverse transcriptase (RT).
• To assess the impact of site-directed mutagenesis, specifically the Asp443 → Ala mutation, on enzyme activity and stability.
• To evaluate the potential of these enhanced HIV-1 RT enzymes for complementary DNA (cDNA) synthesis.

Main Methods:

• Site-directed mutagenesis was employed to introduce the Asp443 → Ala mutation into the p66 subunit of HIV-1 M RT and HIV-1 O RT, abolishing RNase H activity.
• Thermostability was determined by measuring the temperature at which 50% of initial enzyme activity was lost.
• Complementary DNA (cDNA) synthesis activity was assessed at various temperatures to identify the optimal range for the engineered enzymes.

Main Results:

• The engineered HIV-1 M p66D443A/p51 and HIV-1 O p66D443A/p51 mutants exhibited increased thermostability compared to their wild-type counterparts.
• The temperatures reducing 50% activity for the mutants were 44 °C (HIV-1 M) and 52 °C (HIV-1 O), higher than wild-types (42 °C and 48 °C, respectively).
• Both engineered mutants demonstrated sustained cDNA synthesis activity up to 68 °C, surpassing the wild-type enzymes' activity limits (62 °C and 66 °C).

Conclusions:

• The Asp443 → Ala mutation significantly enhances the thermostability of both HIV-1 M and HIV-1 O reverse transcriptase.
• These thermostable HIV-1 RT mutants retain their enzymatic activity at higher temperatures, making them potentially superior tools for cDNA synthesis.
• The engineered enzymes offer improved performance characteristics for molecular biology techniques requiring robust reverse transcriptase activity.