Two avian sarcoma virus mutants with defects in the DNA polymerase-RNase H complex

Area of Science:

• Virology
• Molecular Biology
• Enzymology

Background:

• Avian sarcoma virus (ASV) is a retrovirus encoding an RNA-dependent DNA polymerase (reverse transcriptase) essential for its replication.
• Mutants of ASV provide valuable tools for dissecting the functions of viral proteins, including reverse transcriptase.
• Understanding the enzymatic properties of reverse transcriptase is crucial for comprehending viral replication mechanisms.

Purpose of the Study:

• To characterize the RNA-dependent DNA polymerase and RNase H activities of two distinct ASV mutants, LA 338 and LA 672.
• To investigate the relationship between enzyme properties and viral replication phenotypes.
• To elucidate the molecular basis for replication defects in ASV mutants.

Main Methods:

• Purification of RNA-dependent DNA polymerase-RNase H complex from wild-type and mutant ASV strains.
• Assays for DNA polymerase and RNase H enzymatic activity.
• Thermostability studies of purified enzymes.
• Analysis of enzyme subunit composition and protein levels using polyacrylamide gel electrophoresis and immunological assays.

Main Results:

• The LA 338 mutant exhibited a twofold more thermolabile RNA-dependent DNA polymerase-RNase H complex compared to wild-type ASV. Its enzyme showed rapid loss of response to synthetic template-primers, with no protection from inactivation.
• The LA 672 mutant displayed a late-acting replication block. Its purified enzyme was not thermolabile but showed a 20-fold reduced specific activity in both DNA polymerase and RNase H functions.
• Noninfectious progeny from LA 672 contained immunologically detectable reverse transcriptase with approximately twofold lower levels of alpha and beta subunits.

Conclusions:

• The two ASV mutants, LA 338 and LA 672, possess distinct defects in their RNA-dependent DNA polymerase and RNase H activities, correlating with their different replication phenotypes.
• LA 338's mutation likely affects enzyme stability or substrate interaction, leading to impaired polymerase function.
• LA 672's mutation appears to impact enzyme function or stability post-translationally, resulting in reduced specific activity and replication defects, despite seemingly normal protein levels.