Dataset Information

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

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.

SUBMITTER: Huet S

PROVIDER: S-EPMC2812328 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Huet Sébastien S Avilov Sergiy V SV Ferbitz Lars L Daigle Nathalie N Cusack Stephen S Ellenberg Jan J

Journal of virology 20091111 3

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 strai ...[more]

PMID: 19906916

Similar Datasets

Project description:Study of the density, spatial distribution, and molecular interactions of receptors on the cell membrane provides the knowledge required to understand cellular behavior and biological functions, as well as to discover, design, and screen novel therapeutic agents. However, the mapping of receptor distribution and the monitoring of ligand-receptor interactions on live cells in a spatially and temporally ordered manner are challenging tasks. In this paper, we apply fluorescence correlation spectroscopy (FCS) to map receptor densities on live cell membranes by introducing fluorescently marked aptamer molecules, which specifically bind to certain cell-surface receptors. The femtoliter-sized (0.4 fL) observation volume created by FCS allows fluorescent-aptamer detection down to 2 molecules and appears to be an ideal and highly sensitive biophysical tool for studying molecular interactions on live cells. Fluorophore-labeled aptamers were chosen for receptor recognition because of their high binding affinity and specificity. Aptamer sgc8, generated for specific cell recognition by a process called cell systematic evolution of ligands by exponential enrichment, was determined by FCS to have a binding affinity in the picomolar range (dissociation constant K(d)=790+/-150 pM) with its target membrane receptor, human protein tyrosine kinase-7 (PTK7), a potential cancer biomarker. We then constructed a cellular model and applied this aptamer-receptor interaction to estimate receptor densities and distributions on the cell surface. Specifically, different expression levels of PTK7 were studied by using human leukemia CCRF-CEM cells (1300+/-190 receptors microm(-2)) and HeLa cervical cancer cells (550+/-90 receptors microm(-2)). Competition studies with excess nonlabeled aptamers and proteinase treatment studies proved the validity of the density-estimation approach. With its intrinsic advantages of direct measurement, high sensitivity, fast analysis, and single-cell measurement, this FCS density-estimation approach holds potential for future applications in molecular-interaction studies and density estimations for subcellular structures and membrane receptors.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets