tetano
Editor, Senior Moderator
J Virol. 2011 Nov 23. [Epub ahead of print]
Replication competent influenza A virus encoding split-GFP tagged PB2 polymerase subunit allows live cell imaging of the viral life cycle.
Avilov SV, Moisy D, Munier S, Schraidt O, Naffakh N, Cusack S.
Source
European Molecular Biology Laboratory, Grenoble Outstation, 6 rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France.
Abstract
Studies on the intracellular trafficking of influenza virus ribonucleoproteins are currently limited by the lack of a method enabling their visualisation during infection in single cells. This is largely due to the difficulty to encode fluorescent fusion proteins within the viral genome. To circumvent this limitation, we used the split-GFP system (Cabantous et al., 2005) to produce a quasi-wild-type recombinant A/WSN/33/influenza virus which allows expression of individually fluorescent PB2 polymerase subunits in infected cells. The viral PB2 proteins were fused to the 16 C-terminal amino acids of the GFP, whereas the large trans-complementing GFP fragment was supplied by transient or stable expression in cultured cells that were permissive to infection. This system was used to characterize the intranuclear dynamics of PB2 by fluorescence correlation spectroscopy and to visualize the trafficking of viral ribonucleoproteins (vRNPs) by dynamic light microscopy, in live infected cells. Following nuclear export, vRNPs showed a transient pericentriolar accumulation, and intermittent rapid (∼1 μm/s) directional, movements in the cytoplasm dependent on both microtubules and actin filaments. Our data establish the potential of split-GFP-based recombinant viruses for the tracking of viral proteins during a quasi wild-type infection. This new virus, or adaptations of it, will be of use in elucidating many aspects of influenza virus host cell interactions as well as in screening for new anti-viral compounds. Furthermore, the existence of cell lines stably expressing the complementing GFP fragment will facilitate applications to many other viral and non-viral systems.
PMID:
22114331
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/22114331
Replication competent influenza A virus encoding split-GFP tagged PB2 polymerase subunit allows live cell imaging of the viral life cycle.
Avilov SV, Moisy D, Munier S, Schraidt O, Naffakh N, Cusack S.
Source
European Molecular Biology Laboratory, Grenoble Outstation, 6 rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France.
Abstract
Studies on the intracellular trafficking of influenza virus ribonucleoproteins are currently limited by the lack of a method enabling their visualisation during infection in single cells. This is largely due to the difficulty to encode fluorescent fusion proteins within the viral genome. To circumvent this limitation, we used the split-GFP system (Cabantous et al., 2005) to produce a quasi-wild-type recombinant A/WSN/33/influenza virus which allows expression of individually fluorescent PB2 polymerase subunits in infected cells. The viral PB2 proteins were fused to the 16 C-terminal amino acids of the GFP, whereas the large trans-complementing GFP fragment was supplied by transient or stable expression in cultured cells that were permissive to infection. This system was used to characterize the intranuclear dynamics of PB2 by fluorescence correlation spectroscopy and to visualize the trafficking of viral ribonucleoproteins (vRNPs) by dynamic light microscopy, in live infected cells. Following nuclear export, vRNPs showed a transient pericentriolar accumulation, and intermittent rapid (∼1 μm/s) directional, movements in the cytoplasm dependent on both microtubules and actin filaments. Our data establish the potential of split-GFP-based recombinant viruses for the tracking of viral proteins during a quasi wild-type infection. This new virus, or adaptations of it, will be of use in elucidating many aspects of influenza virus host cell interactions as well as in screening for new anti-viral compounds. Furthermore, the existence of cell lines stably expressing the complementing GFP fragment will facilitate applications to many other viral and non-viral systems.
PMID:
22114331
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/22114331