Abstract
The influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. As PB2 is a relevant host-range determinant we expressed a TAP-tagged PB2 in human cells and isolated intracellular complexes. Alpha-importin was identified as a PB2-associated factor by proteomic analyses. To study the relevance of this interaction for virus replication we mutated the PB2 NLS and analysed the phenotype of mutant subunits, polymerase complexes and RNPs. While mutant PB2 proteins showed reduced nuclear accumulation, they formed polymerase complexes normally when co expressed with PB1 and PA. However, mutant RNPs generated with a viral CAT replicon showed up to hundred-fold reduced CAT accumulation. Rescue of nuclear localisation of mutant PB2 by insertion of an additional SV40 TAg-derived NLS did not revert the mutant phenotype of RNPs. Furthermore, determination of recombinant RNP accumulation in vivo indicated that PB2 NLS mutations drastically reduced virus RNA replication. These results indicate that, above and beyond its role in nuclear accumulation, PB2 interaction with α-importins is required for virus RNA replication. To ascertain whether PB2-α-importin binding could contribute to the adaptation of H5N1 avian viruses to man, their association in vivo was determined. Human alpha importin isoforms associated efficiently to PB2 protein of an H3N2 human virus but bound to diminished and variable extents to PB2 from H5N1 avian or human strains, suggesting that the function of alpha importin during RNA replication is important for the adaptation of avian viruses to the human host.
Highlights
Transcription and replication of influenza A virus is carried out in the nucleus of the infected cells by each of the eight ribonucleoprotein particles (RNPs) that constitute their genome
It was not surprising to identify a-importin among the proteins associated to PB2 (Fig. 1), since a nuclear localisation signals (NLSs) has been identified at its C-terminus [7] and a PB2 C-terminal domain could be co-crystallised with a-importin [10]
In agreement with previous data reported for NLS domain-a-importin interaction, mutation of the NLS resulted in defective nuclear import of mutant PB2 proteins, none of the mutations led to exclusion of the protein from the nucleus (Fig. 2B) and their capacity to form polymerase complexes in vivo was not altered (Fig. 3)
Summary
Transcription and replication of influenza A virus is carried out in the nucleus of the infected cells by each of the eight ribonucleoprotein particles (RNPs) that constitute their genome (reviewed in [1,2,3]). Each RNP contains one single-stranded RNA segment encapsidated by binding to nucleoprotein (NP) monomers and the polymerase complex [4], a heterotrimer containing the PB1, PB2 and PA subunits. The PB1 subunit is responsible for the polymerase and endonuclease activities, PB2 binds the cap structure of cellular pre-mRNAs and PA is a phosphoprotein with protease activity involved in RNA replication (reviewed in [1,2]). These subunits form a tight and stable complex whose low-resolution structure has been determined by electron microscopy and image processing [5,6]. Alternative proposals are based on the formation of Hsp complexes with PB1 and PB2 and suggest the formation of PB1-PB2 and PB1-PA dimers and their co-transport to the nucleus [14,15]
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