Nuclear Import and Assembly of Influenza A Virus RNA Polymerase Studied in Live Cells by Fluorescence Cross-Correlation Spectroscopy

Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Journal of Virology (Impact Factor: 4.44). 11/2009; 84(3):1254-64. DOI: 10.1128/JVI.01533-09
Source: PubMed


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.

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Available from: Sergiy V Avilov
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    • "The component proteins of vRNPs, PB2, PB1, PA and NP contain an NLS within their sequences [21], [22], [23], [24]. PB2 is independently transported to the nucleus, whereas PB1 and PA form a dimer in the cytoplasm, which is then transported into the nucleus [25]. NPs are major components of vRNPs, which are bound to vRNA at a distance of 24 nucleotides [26], [27]. "
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    ABSTRACT: The nucleoprotein (NP) of influenza A virus is transported into the nucleus via the classical importin α/β pathway, and proceeds via nuclear localization signals (NLSs) recognized by importin α molecules. Although NP binds to importin α isoforms Rch1, Qip1 and NPI-1, the role of each individual isoform during the nuclear transport of NP and replication of the influenza virus remains unknown. In this study, we examined the contribution of importin α isoforms for nuclear localization of NP and viral growth using a panel of NP mutants containing serial alanine replacements within an unconventional NLS of NP. Alanine mutation at amino acid 8 (R8A) caused a significant reduction in the nuclear localization and binding to the three importin isoforms. The R8A NP mutant virus did not generate by reverse-genetics approach. This indicates that position 8 is the main site that mediates nuclear localization via interactions with Rch1, Qip1 and NPI-1, and subsequent viral production. This was confirmed by the finding that the conservation of amino acid 8 in human- and avian-origin influenza virus NP was necessary for virus propagation. By contrast, another mutant, S9A NP, which localized in the nucleus, caused a reduction in viral growth and vRNA transcription, suggesting that the unconventional NLS within NP may be associated with nuclear transport, vRNA transcription and viral replication through independent pathways. Interestingly, the N-terminal 110-amino acid region, which contained the unconventional NLS with S9A mutation, mainly bound to Qip1. Furthermore, activities of vRNA transcription and replication of S9A NP mutants were decreased by silencing Qip1 in without changing nuclear localization, indicating that Qip1 involves in multiplication of S9A mutant virus independently of nuclear transport function. Collectively, our results demonstrate the unconventional NLS within NP might have the additional ability to regulate the viral replication that is independent of nuclear localization activity via interactions with Qip1.
    Full-text · Article · Sep 2013 · PLoS ONE
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    • "Significant new insights could come from the direct observation of viral components in live infected cells by advanced fluorescence microscopy techniques. Live cell imaging is commonly performed with transiently co-expressed influenza proteins (e.g., [3] [4] [5]) or with a plasmid-based " minireplicon " system composed of synthetic viral RNAs, NP and three viral polymerase subunits [6]. Unfortunately , these systems do not model the time-dependent changes of composition and localization of viral proteins and ribonucleoproteins , nor the modified cellular context, which occur during a real infection cycle. "
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    ABSTRACT: Dynamic studies of influenza virus infection in the live cells are limited because of the lack of appropriate methods for non-invasive detection of the viral components. Using the split-GFP strategy, we have recently developed and characterized an unimpaired recombinant influenza A virus encoding a tagged PB2 subunit of RNA-dependent RNA polymerase, which enabled continuous real-time visualization of the viral ribonucleoproteins (vRNPs) in living cells (Avilov, Moisy, Munier, Schraidt, Naffakh and Cusack [12]). Here, using this virus, we studied vRNP trafficking and interaction with Rab11 in the context of quasi-wild type infection. In agreement with recent reports, we observed that upon nuclear export, progeny vRNPs accumulate in the particles containing Rab11, a multifunctional protein involved in vesicle trafficking which resides at recycling endosomes. Fluorescence resonance energy transfer microscopy indicated a distance <10nm between PB2 and Rab11, suggesting that a direct interaction occurs. Single particle tracking analysis showed that most of the motions of vRNP-positive particles in infected cells are slow, while rapid directional motions intermittently occur. Analysis focused on these intermittent motions indicated that depolymerization of either microtubules or actin filaments moderately reduced their occurrence, while disruption of both cytoskeleton components in combination suppressed the rapid motions entirely. Thus, the split-GFP based virus enabled us to obtain a live-cell based confirmation for the model of vRNP trafficking which assumes accumulation of vRNP in recycling endosomes through a direct interaction of PB2 with Rab11, and subsequent transport across the cytoplasm involving microtubules and actin filaments.
    Full-text · Article · Oct 2012 · Vaccine
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    • "Previous studies showed that PA-PB1 complex formation is required for the nuclear translocation of both proteins (Fodor and Smith, 2004). PB1 also interacts with PB2 (Fig. 1), which translocates to nuclei by itself, but the effect of a PB1-PB2 interaction on nuclear translocation of PB1 has not been clearly identified, except in studies using tagged proteins (Huet et al., 2010). To determine the effects of co-expressed PA and PB2 on nuclear translocation of PB1, we expressed PB1 fused with eGFP at its Cterminus (PB1eGFP) together with PA or PB2 and visualized the localization of the proteins by fluorescent microscopy (Fig. 3). "
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    ABSTRACT: The influenza polymerase complex composed of PA, PB1 and PB2, plays a key role in viral replication and pathogenicity. Newly synthesized components must be translocated to the nucleus, where replication and transcription of viral genomes take place. Previous studies suggest that while PB2 is translocated to the nucleus independently, PA and PB1 subunits could not localize to the nucleus unless in a PA-PB1 complex. To further determine the molecular interactions between the components, we created a panel of 16 hybridoma cell lines, which produce monoclonal antibodies (mAbs) against each polymerase component. We showed that, although PB1 interacts with both PA and PB2 individually, nuclear localization of PB1 is enhanced only when co-expressed with PA. Interestingly, one of the anti-PA mAbs reacted much more strongly with PA when co-expressed with PB1. These results suggest that PA-PB1 interactions induce a conformational change in PA, which could be required for its nuclear translocation.
    Full-text · Article · Apr 2012 · Virology
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