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

Nuclear Import and Assembly of Influenza A Virus RNA Polymerase Studied in Live Cells by... Nuclear Import and Assembly of Influenza A Virus RNA Polymerase Studied in Live Cells by Fluorescence Cross-Correlation Spectroscopy ▿ † Sébastien Huet 1 , Sergiy V. Avilov 1 , 2 , 3 , Lars Ferbitz 2 , ‡ , Nathalie Daigle 1 , Stephen Cusack 2 , * and Jan Ellenberg 1 , * 1 Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany 2 Grenoble Outstation, European Molecular Biology Laboratory, 6 rue Jules Horowitz, 38042 Grenoble cedex 9, France 3 Palladin Institute of Biochemistry, 9 Leontovich str., Kiev 01030, Ukraine ABSTRACT 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Virology American Society For Microbiology

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

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

Journal of Virology , Volume 84 (3): 1254 – Feb 1, 2010

Abstract

Nuclear Import and Assembly of Influenza A Virus RNA Polymerase Studied in Live Cells by Fluorescence Cross-Correlation Spectroscopy ▿ † Sébastien Huet 1 , Sergiy V. Avilov 1 , 2 , 3 , Lars Ferbitz 2 , ‡ , Nathalie Daigle 1 , Stephen Cusack 2 , * and Jan Ellenberg 1 , * 1 Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany 2 Grenoble Outstation, European Molecular Biology Laboratory, 6 rue Jules Horowitz, 38042 Grenoble cedex 9, France 3 Palladin Institute of Biochemistry, 9 Leontovich str., Kiev 01030, Ukraine ABSTRACT 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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References (54)

Publisher
American Society For Microbiology
Copyright
Copyright © 2010 by the American society for Microbiology.
ISSN
0022-538X
eISSN
1098-5514
DOI
10.1128/JVI.01533-09
pmid
19906916
Publisher site
See Article on Publisher Site

Abstract

Nuclear Import and Assembly of Influenza A Virus RNA Polymerase Studied in Live Cells by Fluorescence Cross-Correlation Spectroscopy ▿ † Sébastien Huet 1 , Sergiy V. Avilov 1 , 2 , 3 , Lars Ferbitz 2 , ‡ , Nathalie Daigle 1 , Stephen Cusack 2 , * and Jan Ellenberg 1 , * 1 Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany 2 Grenoble Outstation, European Molecular Biology Laboratory, 6 rue Jules Horowitz, 38042 Grenoble cedex 9, France 3 Palladin Institute of Biochemistry, 9 Leontovich str., Kiev 01030, Ukraine ABSTRACT 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.

Journal

Journal of VirologyAmerican Society For Microbiology

Published: Feb 1, 2010

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