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 ⌬P, 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.Rabies virus (RV) of the Lyssavirus genus and related members of the Rhabdoviridae, such as vesicular stomatitis virus (VSV, Vesiculovirus genus), are composed of only five multifunctional viral proteins, namely, nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and a large (L) RNA-dependent RNA polymerase. The viral RNA is enwrapped with N and associated with P and L to form a typical helical ribonucleoprotein (RNP) complex, which is active in RNA synthesis. During RV assembly, highly condensed RNPs of the typical rhabdovirus or bullet shape are enwrapped into an envelope containing the M and G proteins (31, 32). Entry of rhabdoviruses into cells involves receptor-mediated endocytosis, pH-dependent fusion of the viral and endosomal membranes (21), release into the cytoplasm, and uncoating of RNPs from M (35) such that gene expression can resume. Although these basic principles of the rhabdovirus infection pathway have been known for some time, details of many particular steps during entry, uncoating, gene expression, and virus egress await illumination. The possibility of real-time visualization of the entire infection pathway of rhabdoviruses and of tracking virus components such as RNPs in living cells is one highly desirable tool for the study of the rhabdovirus life cycle. A widely used approach in imaging of proteins or v...

HIV-1–cellular interactions analyzed by single virus tracing

“…With this technique, the individual steps of the entry process and the dynamics of these processes can be elucidated. SVT has been utilized to investigate the entry and trafficking of several viruses including adeno-associated virus (Bräuchle et al 2002;Seisenberger et al 2001), adenovirus (Suomalainen et al 1999), simian virus 40 (Damm et al 2005), murine polyoma virus (Ewers et al 2005), HIV (McDonald et al 2002), influenza virus (Lakadamyali et al 2003;Rust et al 2004), rabies virus (Finke et al 2004) and murine leukemia virus (Lehmann et al 2005). From the trajectories of individual viruses, the interactions between these particles and different cellular components can be directly observed.…”

Transcriptional Activation of Alpha/Beta Interferon Genes: Interference by Nonsegmented Negative-Strand RNA Viruses

“…We have recently identified the RV P protein as a protein that interferes with the activation of IRF-3 (17). Like in other NNSV, the RV P is an essential virus protein that is required for RNA synthesis, in which it acts as a polymerase cofactor, as well as for the assembly of RNPs, in which it (23), and which induced IFN-␤. Similarly, a virus expressing very low amounts of P, just sufficient to support replication of the virus, was a strong IFN inducer.…”

Dynein Light Chain Association Sequences Can Facilitate Nuclear Protein Import

“…Multimodular (DLC-AS-T-agNLS) cassettes were produced using BamHI/BglII cloning as previously described (Rosenkranz et al, 2003) and inserted into mammalian expression plasmids pEPI-GFP or pEGFPC1 fused in frame C-terminal to the coding sequence of green fluorescent protein (GFP; Figure 1). pExpress-dsRed-DLC-1 (dsRed-LC8; Finke et al, 2004) was supplied by K. Conzelmann (Ludwig Maximilians University, Munich, Germany). …”