Nuclear import and assembly of influenza A virus RNA polymerase studied in live cells by fluorescence cross-correlation spectroscopy

J Virol. 2010 Feb;84(3):1254-64. doi: 10.1128/JVI.01533-09. Epub 2009 Nov 11.


Intracellular transport and assembly of the subunits of the heterotrimeric RNA-dependent RNA polymerase constitute a key component of the replication cycle of influenza virus. Recent results suggest that efficient polymerase assembly is a limiting factor in the viability of reassortant viruses. The mechanism of nuclear import and assembly of the three polymerase subunits, PB1, PB2, and PA, is still controversial, yet it is clearly of great significance in understanding the emergence of new strains with pandemic potential. In this study, we systematically investigated the interactions between the polymerase subunits and their localization in living cells by fluorescence cross-correlation spectroscopy (FCCS) and quantitative confocal microscopy. We could show that PB1 and PA form a dimer in the cytoplasm, which is imported into the nucleus separately from PB2. Once in the nucleus, the PB1/PA dimer associates with PB2 to form the trimeric polymerase. Photon-counting histogram analysis revealed that trimeric polymerase complexes can form higher-order oligomers in the nucleus. We furthermore demonstrate that impairing the nuclear import of PB2 by mutating its nuclear localization signal leads to abnormal formation of the trimeric polymerase in the cytoplasm. Taken together, our results demonstrate which of the previously discussed influenza virus polymerase transport models operates in live cells. Our study sheds light on the interplay between the nuclear import of the subunits and the assembly of the influenza virus polymerase and provides a methodological framework to analyze the effects of different host range mutations in the future.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cell Nucleus / enzymology*
  • DNA-Directed RNA Polymerases / metabolism*
  • Humans
  • Influenza A virus / enzymology*
  • Spectrometry, Fluorescence / methods*
  • Subcellular Fractions / enzymology


  • DNA-Directed RNA Polymerases