title: Tracking fluorescence-labeled rabies virus: enhanced green fluorescent protein-tagged phosphoprotein P supports virus gene expression and formation of infectious particles.
abstract: Rhabdoviruses such as rabies virus (RV) encode only five multifunctional proteins accomplishing viral gene expression and virus formation. The viral phosphoprotein, P, is a structural component of the viral ribonucleoprotein (RNP) complex and an essential cofactor for the viral RNA-dependent RNA polymerase. We show here that RV P fused to enhanced green fluorescent protein (eGFP) can substitute for P throughout the viral life cycle, allowing fluorescence labeling and tracking of RV RNPs under live cell conditions. To first assess the functions of P fusion constructs, a recombinant RV lacking the P gene, SAD DeltaP, was complemented in cell lines constitutively expressing eGFP-P or P-eGFP fusion proteins. P-eGFP supported the rapid accumulation of viral mRNAs but led to low infectious-virus titers, suggesting impairment of virus formation. In contrast, complementation with eGFP-P resulted in slower accumulation of mRNAs but similar infectious titers, suggesting interference with polymerase activity rather than with virus formation. Fluorescence microscopy allowed the detection of eGFP-P-labeled extracellular virus particles and tracking of cell binding and temperature-dependent internalization into intracellular vesicles. Recombinant RVs expressing eGFP-P or an eGFP-P mutant lacking the binding site for dynein light chain 1 (DLC1) instead of P were used to track interaction with cellular proteins. In cells expressing a DsRed-labeled DLC1, colocalization of DLC1 with eGFP-P but not with the mutant P was observed. Fluorescent labeling of RV RNPs will allow further dissection of virus entry, replication, and egress under live-cell conditions as well as cell interactions.
authors.affiliation: Max-von-Pettenkofer Institute & Gene Center, Feodor-Lynen-Strasse 25, D-81377 Münich, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Finke
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Brzózka
authors.forename: Karl-Klaus
authors.initials: KK
authors.lastname: Conzelmann
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D005453:Fluorescence; D015870:Gene Expression; D011820:Rabies virus / Q000235:genetics / Q000502:physiology*; D012261:Ribonucleoproteins / Q000502:physiology*; D014764:Viral Proteins / Q000502:physiology*; D014771:Virion / Q000502:physiology*
chemical_list: D012261:Ribonucleoproteins; D014764:Viral Proteins