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Influenza virus endoribonuclease.

K Klumpp¹, L Hooker, B Handa

¹Roche Products Ltd., Welwyn Garden City, Hertfordshire AL7 3AY, United Kingdom.

Methods in Enzymology

|October 6, 2001

Summary

This study details the purification of active influenza virus polymerase and its endonuclease activity. Optimized methods reveal metal ion requirements and substrate binding kinetics for RNA cleavage.

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Area of Science:

Virology
Molecular Biology
Biochemistry

Background:

The influenza virus polymerase complex, comprising PB1, PB2, and PA subunits, possesses endoribonuclease and RNA-dependent RNA polymerase activities.
These enzymatic activities have historically required the complete three-subunit complex for observation.

Purpose of the Study:

To describe a robust procedure for purifying the active influenza virus polymerase complex with genomic RNA and nucleoprotein.
To synthesize capped RNA substrates for studying the influenza virus endonuclease.
To characterize the enzymatic properties of the influenza virus endoribonuclease using a model RNA substrate.

Main Methods:

Purification of the influenza virus polymerase complex from viral particles.
Synthesis of capped RNA molecules (G20 RNA) as endonuclease substrates.
Enzymatic assays under steady-state and single-turnover conditions to determine kinetics and metal ion dependencies.

Main Results:

Optimized conditions allow for the study of the chemical step of RNA cleavage under single turnover.
The endonuclease activity is dependent on divalent metal ions, with Mn(II), Co(II), and Fe(II) being highly efficient.
Slow binding of Zn(II) and Ni(II) suggests conformational changes are necessary for activity with these ions. DOC detergent inhibits activity and disrupts the complex.

Conclusions:

The study provides a refined method for purifying and assaying the influenza virus endonuclease.
Characterization reveals specific metal ion requirements and kinetic properties, including product dissociation limitations.
Understanding these enzymatic activities is crucial for comprehending influenza virus replication and for potential therapeutic target development.

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