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Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells
11:20

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Published on: June 3, 2012

Influenza A virus RNA polymerase subunit PB2 is the endonuclease which cleaves host cell mRNA and functions only as

L Shi1, D F Summers, Q Peng

  • 1Department of Microbiology and Molecular Genetics, University of California, Irvine 92715-4025, USA.

Virology
|April 1, 1995
PubMed

Insights

Influenza A virus uses its PB2 protein subunit to cleave host cell mRNA, creating primers for viral mRNA synthesis. This endonuclease activity requires the full influenza polymerase complex for function.

Area of Science:

  • Virology
  • Molecular Biology
  • Biochemistry

Background:

  • Influenza A virus RNA polymerase is essential for viral transcription and replication.
  • Viral mRNA synthesis initiates with endonucleolytic cleavage of host cell mRNAs to generate capped primers.
  • The precise mechanism and subunit roles in this cap-dependent endonuclease activity remain incompletely understood.

Purpose of the Study:

  • To investigate the properties of the capped RNA-specific endonuclease associated with the influenza virus polymerase.
  • To determine the roles of the individual polymerase subunits in transcription initiation.
  • To elucidate the mechanism of host mRNA cleavage for viral primer synthesis.

Main Methods:

  • Development of an in vitro assay using an artificial cap-0 RNA substrate.
  • Cleavage assays with purified virion ribonucleoprotein complexes.
  • Inhibition studies using specific polyclonal antibodies against polymerase subunits (PB2, PB1, PA).
  • Enzyme activity assays with partially purified trimeric polymerase and its subunit-depleted forms.

Main Results:

  • Purified influenza ribonucleoprotein complexes specifically cleaved the RNA substrate to produce a 14-nt capped RNA fragment.
  • Anti-PB2 IgG inhibited endonuclease activity, while anti-PB1 and anti-PA antibodies did not.
  • The trimeric influenza polymerase complex exhibited cleavage activity.
  • Removal of one or two subunits from the polymerase complex abolished endonuclease activity.

Conclusions:

  • The viral PB2 protein subunit functions as the endonuclease responsible for cleaving host cell mRNA to generate primers.
  • The endonuclease activity of PB2 is dependent on its association with the other two influenza polymerase subunits (PB1 and PA).
  • The complete influenza RNA polymerase complex is necessary for efficient primer generation, highlighting the importance of subunit interactions.

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