Isolation and characterization of Rous sarcoma virus recombinant reverse transcriptase dimers

Area of Science:

• Molecular Biology
• Virology
• Enzymology

Background:

• Reverse transcriptase (RT) is crucial for retroviral replication.
• Understanding the molecular forms and activities of RT is essential for developing antiviral strategies.
• Rous sarcoma virus (RSV) provides a model system for studying retroviral enzymes.

Purpose of the Study:

• To isolate and characterize different molecular forms of RSV reverse transcriptase (RT) expressed in E. coli.
• To compare the enzymatic activities (DNA polymerase and RNase H) of various RT dimer forms.
• To assess the similarity of recombinant RSV RT forms to virion-associated RT.

Main Methods:

• Expression of the Rous sarcoma virus (RSV) pol gene in E. coli using plasmid pMF14.
• Isolation and purification of different RT molecular forms (alphaalpha, betabeta, alphabeta dimers).
• Enzymatic assays to determine DNA polymerase and RNase H activities of purified RT forms.

Main Results:

• Three dimeric forms of RSV RT (alphaalpha, betabeta, alphabeta) were successfully isolated.
• All three forms exhibited DNA polymerase activity, with relative activities of 1:3:4 for alphaalpha, betabeta, and alphabeta, respectively.
• RNase H activity was present in betabeta and alphabeta dimers but absent in the alphaalpha dimer.

Conclusions:

• The study successfully produced and characterized recombinant RSV RT forms in E. coli.
• The differential enzymatic activities of the RT dimers provide insights into the functional roles of subunits.
• The findings suggest that the recombinant betabeta and alphabeta RSV RT dimers closely mimic their native counterparts found in virions.