Recombinant HIV-1 reverse transcriptase: purification, primary structure, and polymerase/ribonuclease H activities

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Human Immunodeficiency Virus type 1 (HIV-1) reverse transcriptase (RT) is a crucial enzyme for viral replication.
• Production of active and pure recombinant HIV-1 RT is essential for biochemical studies and therapeutic development.

Purpose of the Study:

• To develop a stable and efficient expression system for recombinant HIV-1 RT in Escherichia coli.
• To characterize the enzymatic activity and subunit composition of the expressed HIV-1 RT.

Main Methods:

• Utilized a double-plasmid expression system in E. coli for co-expression of HIV-1 RT precursor and HIV-1 protease.
• Purified the recombinant enzyme to >90% homogeneity, consisting of p66 and p51 subunits.
• Assessed enzymatic activity using polymerase and RNase H assays, and inhibitor sensitivity with phosphonoformate.

Main Results:

• Successfully produced soluble, active recombinant HIV-1 RT comprising equimolar p66 and p51 subunits with a common N-terminus.
• The recombinant enzyme exhibited specific activity comparable to native viral RT and identical sensitivity to phosphonoformate.
• RNase H activity was exclusively localized to the p66 subunit, supporting its C-terminal domain location.

Conclusions:

• The established E. coli expression system provides a reliable method for producing functional HIV-1 RT.
• The recombinant enzyme is biochemically similar to the native viral enzyme, suitable for further research.
• The p66 subunit contains the RNase H activity, crucial for understanding HIV-1 replication mechanisms.