Purification and partial characterization of equine infectious anemia virus reverse transcriptase

Area of Science:

• Virology
• Molecular Biology
• Immunology

Background:

• The C-terminal region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a target for antibody C2003.
• This RT sequence is conserved in equine infectious anemia virus (EIAV), a related lentivirus.
• Antibody C2003 previously showed cross-reactivity with native EIAV RT and inhibited its DNA polymerase activity.

Purpose of the Study:

• To further characterize equine infectious anemia virus reverse transcriptase (EIAV RT).
• To investigate the structural and functional properties of EIAV RT using antibody-based methods.
• To compare EIAV RT with other viral reverse transcriptases, particularly HIV-1 RT.

Main Methods:

• Immunoaffinity chromatography using immobilized C2003 antibody to fractionate EIAV RT.
• In situ activity gel assays to determine the enzymatic activity of EIAV RT components.
• Fidelity assays of DNA synthesis by EIAV RT.

Main Results:

• EIAV RT was successfully fractionated into an equimolar mixture of 66 kDa and 51 kDa proteins.
• Both DNA polymerase and RNase H activities were associated with these two protein components.
• Polymerase activity was primarily linked to the 66 kDa EIAV RT component.
• The fidelity of DNA synthesis by EIAV RT was comparable to HIV-1 RT and lower than avian myeloblastosis virus (AMV) RT.

Conclusions:

• EIAV RT exists as a heterodimer composed of 66 kDa and 51 kDa subunits.
• The enzymatic activities of EIAV RT are associated with these subunits.
• EIAV RT shares significant structural and functional similarities with HIV-1 RT.