Membrane Accumulation of Influenza A Virus Hemagglutinin Triggers Nuclear Export of the Viral Genome via Protein Kinase Cα-mediated Activation of ERK Signaling

doi:10.1074/jbc.M510233200

Membrane Accumulation of Influenza A Virus Hemagglutinin Triggers Nuclear Export of the Viral Genome via Protein Kinase Cα-mediated Activation of ERK Signaling*

^‡Institute for Medical Virology, Justus-Liebig-University, Frankfurter Strasse 107, D-35392 Giessen, the ^¶Institute for Virology, Philipps University, Robert-Koch Strasse 17, D-35037 Marburg, the ^∥Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Institute for Immunology, Paul-Ehrlich Strasse 28, D-72076 Tübingen, and the ^§Institute for Molecular Virology, Westfälische-Wilhelms-University, von-Esmarch Strasse 56, D-48149 Münster, Germany

2 To whom correspondence should be addressed: Tel.: 49-(0)641-99-47750; Fax: 49-(0)641-99-41209; E-mail: stephan.pleschka{at}mikro.bio.uni-giessen.de.

Abstract

Replication and transcription of the influenza virus genome takes place exclusively within the nucleus of the infected cells. The viral RNA genome, polymerase subunits, and nucleoprotein form ribonucleoprotein (RNP) complexes. Late in the infectious cycle RNPs have to be exported from the nucleus to be enwrapped into budding progeny virions at the cell membrane. This process requires viral activation of the cellular Raf/MEK/ERK (mitogen-activated protein kinase (MAPK)) signaling cascade that is activated late in the infection cycle. Accordingly, block of the cascade results in retardation of RNP export and reduced titers of progeny virus. In the present study we have analyzed the importance of cell-membrane association of the viral hemagglutinin glycoprotein for viral MAPK activation. We show that hemagglutinin membrane accumulation and its tight association with lipid-raft domains trigger activation of the MAPK cascade via protein kinase Cα activation and induces RNP export. This may represent an auto-regulative mechanism that coordinates timing of RNP export to a point when all viral components are ready for virus budding.

Footnotes

↵3 The abbreviations used are: MEK, MAPK/ERK kinase; ts, temperature-sensitive; wt, wild type; HA, hemagglutinin; MCD, methyl-β-cyclodextrin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PKC, calcium-dependent protein kinase; RNP, ribonucleoprotein; NP, nucleoprotein; MDCK, Madin-Darby canine kidney cells; PBS, phosphate-buffered saline; m.o.i., multiplicity of infection; TPA, 12-O-tetradecanoylphorbol-13-acetate; IFN, interferon; JNK, c-Jun NH₂-terminal kinase; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorting; Ct-B, choleratoxin B subunit; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; ss, single strand; ds, double strand; wt, wild type; p.i., post infection; p.t., post transfection; dn, dominant negative; GM1, gangliotetraosylceramide.
↵* This work was supported by grants of the Deutsche Forschungsgemeinschaft (Grants IIIGK-GRK370/3 and SFB 535 to S. P., SFB 593 to H.-D. K., and Lu 477/4-5 to S. L.). This work is part of the activities of the VIRGIL European Network of Excellence on Antiviral Drug Resistance supported by the Priority 1 Life Sciences, Genomics and Biotechnology for Health program in the 6th Framework Program of the EU (Grant LSHM-CT-2004-503359). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 Present address: Paul-Ehrlich-Institute, Paul-Ehrlich Strasse 51-59, D-63225 Langen, Germany.
- Received September 16, 2005.
- Revision received March 29, 2006.
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