Visualization of an Alphaherpesvirus Membrane Protein That Is Essential for Anterograde Axonal Spread of Infection in Neurons

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 neur...

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Autores principales:	M. P. Taylor, T. Kramer, M. G. Lyman, R. Kratchmarov, L. W. Enquist
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2012
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/409daa9887be42a39896df1e90fff469
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spelling	oai:doaj.org-article:409daa9887be42a39896df1e90fff4692021-11-15T15:39:09ZVisualization of an Alphaherpesvirus Membrane Protein That Is Essential for Anterograde Axonal Spread of Infection in Neurons10.1128/mBio.00063-122150-7511https://doaj.org/article/409daa9887be42a39896df1e90fff4692012-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00063-12https://doaj.org/toc/2150-7511ABSTRACT 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.M. P. TaylorT. KramerM. G. LymanR. KratchmarovL. W. EnquistAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 3, Iss 2 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 M. P. Taylor T. Kramer M. G. Lyman R. Kratchmarov L. W. Enquist Visualization of an Alphaherpesvirus Membrane Protein That Is Essential for Anterograde Axonal Spread of Infection in Neurons
description	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.
format	article
author	M. P. Taylor T. Kramer M. G. Lyman R. Kratchmarov L. W. Enquist
author_facet	M. P. Taylor T. Kramer M. G. Lyman R. Kratchmarov L. W. Enquist
author_sort	M. P. Taylor
title	Visualization of an Alphaherpesvirus Membrane Protein That Is Essential for Anterograde Axonal Spread of Infection in Neurons
title_short	Visualization of an Alphaherpesvirus Membrane Protein That Is Essential for Anterograde Axonal Spread of Infection in Neurons
title_full	Visualization of an Alphaherpesvirus Membrane Protein That Is Essential for Anterograde Axonal Spread of Infection in Neurons
title_fullStr	Visualization of an Alphaherpesvirus Membrane Protein That Is Essential for Anterograde Axonal Spread of Infection in Neurons
title_full_unstemmed	Visualization of an Alphaherpesvirus Membrane Protein That Is Essential for Anterograde Axonal Spread of Infection in Neurons
title_sort	visualization of an alphaherpesvirus membrane protein that is essential for anterograde axonal spread of infection in neurons
publisher	American Society for Microbiology
publishDate	2012
url	https://doaj.org/article/409daa9887be42a39896df1e90fff469
work_keys_str_mv	AT mptaylor visualizationofanalphaherpesvirusmembraneproteinthatisessentialforanterogradeaxonalspreadofinfectioninneurons AT tkramer visualizationofanalphaherpesvirusmembraneproteinthatisessentialforanterogradeaxonalspreadofinfectioninneurons AT mglyman visualizationofanalphaherpesvirusmembraneproteinthatisessentialforanterogradeaxonalspreadofinfectioninneurons AT rkratchmarov visualizationofanalphaherpesvirusmembraneproteinthatisessentialforanterogradeaxonalspreadofinfectioninneurons AT lwenquist visualizationofanalphaherpesvirusmembraneproteinthatisessentialforanterogradeaxonalspreadofinfectioninneurons
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Visualization of an Alphaherpesvirus Membrane Protein That Is Essential for Anterograde Axonal Spread of Infection in Neurons

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