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In vitro analysis of transneuronal spread of an alphaherpesvirus infection in peripheral nervous system neurons.

ABSTRACT: The neurotropic alphaherpesviruses invade and spread in the nervous system in a directional manner between synaptically connected neurons. Until now, this property has been studied only in living animals and has not been accessible to in vitro analysis. In this study, we describe an in vitro system in which cultured peripheral nervous system neurons are separated from their neuron targets by an isolator chamber ring. Using pseudorabies virus (PRV), an alphaherpesvirus capable of transneuronal spread in neural circuits of many animals, we have recapitulated in vitro all known genetic requirements for retrograde and anterograde transneuronal spread as determined previously in vivo. We show that in vitro transneuronal spread requires intact axons and the presence of the viral proteins gE, gI, and Us9. We also show that transneuronal spread is dependent on the viral glycoprotein gB, which is required for membrane fusion, but not on gD, which is required for extracellular spread. We demonstrate ultrastructural differences between anterograde- and retrograde-traveling virions. Finally, we show live imaging of dynamic fluorescent virion components in axons and postsynaptic target neurons.

SUBMITTER: Feierbach B

PROVIDER: S-EPMC1933274 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ABSTRACT Alphaherpesvirus particles travel long distances in the axons of neurons using host microtubule molecular motors. The transport dynamics of individual virions in neurons have been assessed in cultured neurons, but imaging studies of single particles in tissue from infected mice have not been reported. We developed a protocol to image explanted, infected peripheral nervous system (PNS) ganglia and associated innervated tissue from mice infected with pseudorabies virus (PRV). This ex vivo preparation allowed us to visualize and track individual virions over time as they moved from the salivary gland into submandibular ganglion neurons of the PNS. We imaged and tracked hundreds of virions from multiple mice at different time points. We quantitated the transport velocity, particle stalling, duty cycle, and directionality at various times after infection. Using a PRV recombinant that expressed monomeric red fluorescent protein (mRFP)-VP26 (red capsid) and green fluorescent protein (GFP)-Us9 (green membrane protein), we corroborated that anterograde transport in axons occurs after capsids are enveloped. We addressed the question of whether replication occurs initially in the salivary gland at the site of inoculation or subsequently in the neurons of peripheral innervating ganglia. Our data indicate that significant amplification of infection occurs in the peripheral ganglia after transport from the site of infection and that these newly made particles are transported back to the salivary gland. It is likely that this reseeding of the infected gland contributes to massive invasion of the innervating PNS ganglia. We suggest that this "round-trip" infection process contributes to the characteristic peripheral neuropathy of PRV infection. IMPORTANCE Much of our understanding of molecular mechanisms of alphaherpesvirus infection and spread in neurons comes from studying cultured primary neurons. These techniques enabled significant advances in our understanding of the viral and neuronal components needed for efficient replication and directional spread between cells. However, in vitro systems cannot recapitulate the environment of innervated tissue in vivo with associated defensive properties, such as innate immunity. Therefore, in this report, we describe a system to image the progression of infection by single virus particles in tissue harvested from infected animals. We explanted intact innervated tissue from infected mice and imaged fluorescent virus particles in infected axons of the specific ganglionic neurons. Our measurements of virion transport dynamics are consistent with published in vitro results. Importantly, this system enabled us to address a fundamental biological question about the amplification of a herpesvirus infection in a peripheral nervous system circuit.

Project description:UNLABELLED: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.

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In vitro analysis of transneuronal spread of an alphaherpesvirus infection in peripheral nervous system neurons.

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