Nucleic Acids Res. 2013 Sep;41(17):8289-99.

Characterization of PA-N terminal domain of Influenza A polymerase reveals sequence specific RNA cleavage.

Datta K, Wolkerstorfer A, Szolar OH, Cusack S, Klumpp K.


Hoffmann-La Roche Inc., Virology Discovery, Nutley, NJ 07110, USA, Savira pharmaceuticals GmbH, Veterinaerplatz 1/IA, A-1210, Vienna, Austria, European Molecular Biology Laboratory, Grenoble Outstation, 6 rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France, Unit of Virus Host Cell Interactions, University Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France and RiboScience LLC, 3901 Laguna Avenue, Palo Alto, CA 94306, USA.



Influenza virus uses a unique cap-snatching mechanism characterized by hijacking and cleavage of host capped pre-mRNAs, resulting in short capped RNAs, which are used as primers for viral mRNA synthesis. The PA subunit of influenza polymerase carries the endonuclease activity that catalyzes the host mRNA cleavage reaction. Here, we show that PA is a sequence selective endonuclease with distinct preference to cleave at the 3′ end of a guanine (G) base in RNA. The G specificity is exhibited by the native influenza polymerase complex associated with viral ribonucleoprotein particles and is conferred by an intrinsic G specificity of the isolated PA endonuclease domain PA-Nter. In addition, RNA cleavage site choice by the full polymerase is also guided by cap binding to the PB2 subunit, from which RNA cleavage preferentially occurs at the 12th nt downstream of the cap. However, if a G residue is present in the region of 10-13 nucleotides from the cap, cleavage preferentially occurs at G. This is the first biochemical evidence of influenza polymerase PA showing intrinsic sequence selective endonuclease activity.

PMID: 23847103



The nucleic acid of influenza A virus is a single stranded negative sense RNA occurring as eight segments that remain encapsidated by the viral nucleoprotein (NP). The viral RNA (vRNA) adopts the shape of a panhandle presumably due to base pairing of the 5’ and 3’ end residues of the vRNA.  The encapsidated RNA along with the influenza polymerase form the ribonucleoprotein particles (RNP), which serve as the transcription and replication machinery of the virus (1).

Influenza polymerase comprises of three subunits, PB2 (carrying the cap-binding site) (2), PA (harboring the endonuclease active site) (3) and PB1 (carrying the polymerase active site) (1). To initiate viral transcription, the polymerase employs a unique ‘cap-snatching’ mechanism that involves precise coordination of the three polymerase subunits – a) binding of host capped mRNA at the cap-binding site, b) endonucleolytic cleavage of the phosphodiester bond 10-13 nts downstream from the 5’-cap structure and c) utilizing the capped mRNA segment for priming transcription of full length viral mRNA that is subsequently translated into viral protein (1). Since ‘cap-snatching’ is central to the viral life cycle, the polymerase subunits are ideal targets for development of novel direct acting antivirals.

In this study we investigated whether the host capped mRNA sequence played a role in the initiation of viral transcription. We have shown that the N-terminal domain of the PA subunit of influenza A polymerase exhibits intrinsic guanine (G) specific cleavage, as neither the viral template RNA nor any other polymerase subunits or accessory proteins were essential for such selectivity. Interestingly, in the context of RNP, RNA cleavage was primarily G-specific only when a G residue was present in the 10th -13th nts range downstream of the cap structure of the host mRNA, otherwise cleavage occurred at the 12th position. Based on these findings, the host capped mRNA with a G residue at 10-13th position from the cap moiety appeared to be the preferred substrate for initiation of cap-dependent transcription (4).

While further studies would be required to establish the significance of the observed G specific cleavage, we speculate that the host mRNA with a G in the aforementioned position would enable base pairing with a well conserved C residue at the 3’ end of the vRNA template, thus enhancing optimal primer-template alignment while enabling efficient cleavage at the endonuclease active site. Alternatively, the observed G-specific cleavage may indicate a yet unidentified role of the PA subunit in the influenza A viral life cycle.



  1. Ruigrok, R. W., Crepin, T., Hart, D. J., and Cusack, S. (2010) Current opinion in structural biology 20(1), 104-113
  2. Guilligay, D., Tarendeau, F., Resa-Infante, P., Coloma, R., Crepin, T., Sehr, P., Lewis, J., Ruigrok, R. W., Ortin, J., Hart, D. J., and Cusack, S. (2008) Nature structural & molecular biology 15(5), 500-506
  3. Dias, A., Bouvier, D., Crepin, T., McCarthy, A. A., Hart, D. J., Baudin, F., Cusack, S., and Ruigrok, R. W. (2009) Nature 458(7240), 914-918
  4. Datta K,Wolkerstorfer A., Szolar O., Cusack, S and Klumpp, K. (2013) Nucleic Acids Res. 41(17):8289-8299
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