Double-labeled rabies virus: live tracking of enveloped virus transport.
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.
Klingen
Yvonne
Y
Friedrich-Loeffler-Institute, Boddenblick 5a, D-17493 Greifswald-Riems, Germany.
Conzelmann
Karl-Klaus
KK
Finke
Stefan
S
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D011992:Endosomes / Q000821:virology; D017930:Genes, Reporter; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D018832:Molecular Chaperones; D009474:Neurons / Q000821:virology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011820:Rabies virus / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000737:chemistry; D019065:Virus Assembly / Q000502:physiology; D000097573:Red Fluorescent Protein
D000956:Antigens, Viral; D006023:Glycoproteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D018832:Molecular Chaperones; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins; D015678:Viral Structural Proteins; D000097573:Red Fluorescent Protein; C103961:P phosphoprotein, Rabies virus; C485184:enhanced green fluorescent protein; C064951:glycoprotein G, Rabies virus