Visualization of an Alphaherpesvirus Membrane Protein That Is Essential for Anterograde Axonal Spread of Infection in Neurons
Open Access
- 2 May 2012
- journal article
- research article
- Published by American Society for Microbiology in mBio
- Vol. 3 (2), e00063-12
- https://doi.org/10.1128/mbio.00063-12
Abstract
Pseudorabies virus (PRV), an alphaherpesvirus with a broad host range, replicates and spreads in chains of synaptically connected neurons. The PRV protein Us9 is a small membrane protein that is highly conserved among alphaherpesviruses and is essential for anterograde axonal spread in neurons. Specifically, the Us9 protein is required for the sorting of newly assembled PRV particles into axons. However, the molecular details underlying the function of Us9 are poorly understood. Here we constructed PRV strains that express functional green fluorescent protein (GFP)-Us9 fusion proteins in order to visualize axonal transport of viral particles in infected rat superior cervical ganglion neurons. We show that GFP-Us9-labeled structures are transported exclusively in the anterograde direction within axons. Additionally, the vast majority of anterograde-directed capsids (labeled with VP26-monomeric red fluorescent protein) and a viral membrane protein (labeled with glycoprotein M fused to mCherry) are cotransported with GFP-Us9 in the anterograde direction. In contrast, during infection with PRV strains that express nonfunctional mutant GFP-Us9 proteins, cotransport of mutant GFP-Us9 with capsids in axons is abolished. These findings show that axonal sorting of progeny viral particles is dependent upon the association of viral structures with membranes that contain functional Us9 proteins. This association is required for anterograde spread of infection in neurons. IMPORTANCE Alphaherpesviruses, such as pseudorabies virus (PRV), are parasites of the mammalian nervous system. These viruses spread over long distances in chains of synaptically connected neurons. PRV encodes several proteins that mediate directed virion transport and spread of infection. Us9 is a highly conserved viral membrane protein that is essential for anterograde neuronal spread of infection. In the absence of Us9, newly replicated viral particles are assembled in the cell body but are not sorted into or transported within axons. Here, we constructed and characterized novel PRV strains that express functional green fluorescent protein (GFP)-Us9 fusion proteins in order to visualize its localization in living neurons during infection. This enabled us to better understand the function of Us9 in facilitating the spread of infection. We show that all viral particles moving in the anterograde direction are labeled with GFP-Us9, suggesting that the presence of Us9 determines the capacity for directed transport within axons.Keywords
This publication has 39 references indexed in Scilit:
- Anterograde Transport of Herpes Simplex Virus Capsids in Neurons by both Separate and Married MechanismsJournal of Virology, 2011
- Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virusVirology, 2011
- Ultrastructural Analysis of Virion Formation and Anterograde Intraaxonal Transport of the Alphaherpesvirus Pseudorabies Virus in Primary NeuronsJournal of Virology, 2010
- Anterograde Spread of Herpes Simplex Virus Type 1 Requires Glycoprotein E and Glycoprotein I but Not Us9Journal of Virology, 2009
- Comparison of the Pseudorabies Virus Us9 Protein with Homologs from Other Veterinary and Human AlphaherpesvirusesJournal of Virology, 2009
- Compartmented Neuron Cultures for Directional Infection by Alpha HerpesvirusesCurrent Protocols in Cell Biology, 2009
- Herpes Simplex Virus gE/gI and US9 Proteins Promote Transport of both Capsids and Virion Glycoproteins in Neuronal AxonsJournal of Virology, 2008
- Fusion of Enhanced Green Fluorescent Protein to the Pseudorabies Virus Axonal Sorting Protein Us9 Blocks Anterograde Spread of Infection in Mammalian NeuronsJournal of Virology, 2008
- In Vitro Analysis of Transneuronal Spread of an Alphaherpesvirus Infection in Peripheral Nervous System NeuronsJournal of Virology, 2007
- Viral regulation of the long distance axonal transport of herpes simplex virus nucleocapsidNeuroscience, 2007