Modifications of cellular RNA-polymerase II after infection with frog virus 3

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• RNA-polymerase II is crucial for gene transcription in eukaryotic cells.
• Feline virus 3 (FV3) is a significant pathogen affecting feline populations.
• Viral infections can profoundly impact host cell machinery, including transcription.

Purpose of the Study:

• To investigate the effect of FV3 infection on the functional properties of host cell RNA-polymerase II.
• To compare the binding characteristics of RNA-polymerase II from infected and uninfected cells.

Main Methods:

• Extraction and purification of RNA-polymerase II from both FV3-infected and uninfected BHK cells.
• Assays measuring the binding affinity of RNA-polymerase II for [3-H]-amanitin.
• Determination of apparent Michaelis constants (Km) for ribonucleoside triphosphates.

Main Results:

• FV3 infection significantly altered the binding sites for [3-H]-amanitin on RNA-polymerase II.
• The dissociation constant of the complex between [3H]-amanitin and RNA-polymerase II was significantly modified post-infection.
• Apparent Km values for ribonucleoside triphosphates showed no significant change, indicating unaltered substrate binding.

Conclusions:

• FV3 infection specifically targets and modifies the amanitin-binding properties of RNA-polymerase II.
• The core catalytic function of RNA-polymerase II, as indicated by substrate affinity, is preserved despite FV3 infection.
• These findings suggest a potential mechanism for viral modulation of host transcription distinct from direct catalytic inhibition.