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Journal of Virology, January 2008, p. 237-245, Vol. 82, No. 1
0022-538X/08/$08.00+0     doi:10.1128/JVI.01342-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Double-Labeled Rabies Virus: Live Tracking of Enveloped Virus Transport{triangledown} ,{dagger}

Yvonne Klingen, Karl-Klaus Conzelmann, and Stefan Finke*

Max von Pettenkofer Institute and Gene Center, Ludwig-Maximilians-University Munich, Feodor Lynen Str. 25, D-81377 Munich, Germany

Received 20 June 2007/ Accepted 1 October 2007

Here we describe a strategy to fluorescently label the envelope of rabies virus (RV), of the Rhabdoviridae family, in order to track the transport of single enveloped viruses in living cells. Red fluorescent proteins (tm-RFP) were engineered to comprise the N-terminal signal sequence and C-terminal transmembrane spanning and cytoplasmic domain sequences of the RV glycoprotein (G). Two variants of tm-RFP were transported to and anchored in the cell surface membrane, independent of glycosylation. As shown by confocal microscopy, tm-RFP colocalized at the cell surface with the RV matrix and G protein and was incorporated into G gene-deficient virus particles. Recombinant RV expressing the membrane-anchored tm-RFP in addition to G yielded infectious viruses with mosaic envelopes containing both tm-RFP and G. Viable double-labeled virus particles comprising a red fluorescent envelope and a green fluorescent ribonucleoprotein were generated by expressing in addition an enhanced green fluorescent protein-phosphoprotein fusion construct (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004). Individual enveloped virus particles were observed under live cell conditions as extracellular particles and inside endosomal vesicles. Importantly, double-labeled RVs were transported in the retrograde direction over long distances in neurites of in vitro-differentiated NS20Y neuroblastoma cells. This indicates that the typical retrograde axonal transport of RV to the central nervous system involves neuronal transport vesicles in which complete enveloped RV particles are carried as a cargo.

* Corresponding author. Present address: Friedrich-Loeffler-Institute, Boddenblick 5a, D-17493 Greifswald-Riems, Germany. Phone: 49 38351 7253. Fax: 49 38351 7275. E-mail: stefan.finke{at}fli.bund.de

{triangledown} Published ahead of print on 10 October 2007.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, January 2008, p. 237-245, Vol. 82, No. 1
0022-538X/08/$08.00+0     doi:10.1128/JVI.01342-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Das, S. C., Panda, D., Nayak, D., Pattnaik, A. K. (2009). Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells. J. Virol. 83: 2611-2622 [Abstract] [Full Text]  
  • Marschalek, A., Finke, S., Schwemmle, M., Mayer, D., Heimrich, B., Stitz, L., Conzelmann, K.-K. (2009). Attenuation of Rabies Virus Replication and Virulence by Picornavirus Internal Ribosome Entry Site Elements. J. Virol. 83: 1911-1919 [Abstract] [Full Text]  


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