Imaging Viral Assembly and Trans-synaptic Transport in the Brain

Lynn Enquist, Ph.D.

Princeton University

Funded in December, 2004: $300000 for 4 years
LAY SUMMARY . ABSTRACT . HYPOTHESIS . FINDINGS . SELECTED PUBLICATIONS .

LAY SUMMARY

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Imaging How a Virus is Assembled and Transported Within the Brain

Princeton researchers will image fluorescently labeled viral particles as they pass from one brain neuron to another in the mouse. This will provide information on how viral infection spreads within nervous system.  The study also will demonstrate a novel means for identifying brain cell connections that make up neural networks.  Then scientists could use inactivated viruses to delineate neural networks involved in specific brain functions.

Pseudorabies virus, a type of herpes virus, infects the nervous system by spreading through synaptic connections, rather than being released randomly from infected nerve cells.  The virus, therefore, follows along normal neural networks that perform specific functions.  By fluorescently labeling the viral particles and using two-photon laser scanning to follow their travels along neural networks in mouse tissue and in living mice, the researchers will be able to visualize how the virus is assembled into a nerve cell and how it then moves through nerve cell synapses to spread the infection. Additionally, the researchers will be able to identify specific neural circuits that are revealed by the virus’ path.  

Significance:  This research should provide insight into viral spread in the nervous system and also demonstrate a novel new way to image neural connections in the brain.  Using a non-infectious form of the virus, the technique eventually could be undertaken in humans.

ABSTRACT

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Imaging Viral Assembly and Trans-synaptic Transport in the Brain

α-herpesviruses are remarkable because they are parasites of their natural host's peripheral nervous system. The neuroinvasive attributes of these viruses can be exploited to understand fundamental aspects of the mammalian nervous system. Work in this proposal focuses new imaging technology on mechanisms of movement of virion components inside and between neurons and how such knowledge can be exploited to study neuronal circuitry. This proposal takes advantage of several recent developments: (i) the construction of an extensive set of pseudorabies virus (PRV) fluorescent fusion proteins to facilitate imaging of viral movements, (ii) perfection of multi-chamber tissue culture systems to manipulate and analyze viral spread in vitro, (iii) the development of methods for in vivo Two-photon Laser Scanning Microscopy (TPLSM) of the nervous system, and (iv) the extensive anatomic and cell biological characterization of the mouse submandibular ganglion (SMG).

Aim 1. In vitro analysis of trans-neuronal movements of PRV-infected neurons in a multi-chamber system. Superior cervical ganglion (SCG) neurons from embryonic rats will be cultured in a multi-chamber "Campenot" system and infected with fluorescently-labeled PRV. The multi-chamber system enables the isolation of SCG cell bodies from their axon terminals and the cells to which they connect, thereby separating the site of infection from the site of viral egress. The chambers are readily amenable to manipulation, including electrical stimulation, and can be used for dynamic analysis using living neurons combining fluorescence microscopy (TPLSM, epi-fluorescence) and electrophysiology. In addition, the cells can be fixed for immunofluorescence and electron microscopy.

Aim 2. In vivo analysis of trans-neuronal movements of PRV-infected neurons using the mouse submandibular ganglion (SMG). The SMG has been used for the in vivo imaging of synapses during development and will serve as an in vivo model for viral spread. The SMG will be infected with fluorescently-labeled PRV by injection into the submandibular gland and the spread into and between neuronal cell bodies and presynaptic terminals in the ganglion will be imaged by TPLSM. Electrophysiological recordings and nerve stimulation methods will be used to assess the effects of electrical and synaptic activation on transport.

HYPOTHESIS

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Hypothesis:
The neurotropic α-herpesviruses are common mammalian pathogens that invade the peripheral and central nervous system of their hosts. However, the mechanisms by which they invade and spread in the nervous system are poorly understood. Pseudorabies virus (PRV), a well-studied α-herpesvirus, is capable of spread between synaptically-connected neurons in diverse hosts. This property has been exploited to map a wide variety of neural circuits in many different mammalian species. Despite such progress, we do not understand the mechanism of trans-neuronal spread even in its basic principles. We assert that we can image individual fluorescently-labeled virion components as they assemble, exit, and re-infect living, synaptically connected neurons in vitro and in vivo. We will exploit this new window on viral dynamics to test the hypothesis that PRV crosses between neurons in a synapse-specific manner.

Goals:
In this proposal, high-resolution fluorescence imaging techniques in combination with electrophysiological and pharmacological analyses will elucidate how PRV spreads between living neurons. More specifically, this proposal will address whether PRV undergoes egress at or near synapses. If viral egress is restricted to synapses, then this validates the spatial specificity of PRV as a neural circuit tracer and points to mechanism. Understanding the mechanism by which herpesviruses spread within the nervous system may aid in improving viral neural circuitry tracers and identifying potential therapeutic targets for anti-viral drugs. The aims of this proposal are designed to provide a comprehensive analysis of trans-neuronal viral spread in living neurons and explore a mechanism of spread.

Methods:

In vitro methods:
For in vitro analyses of trans-neuronal spread, superior cervical ganglion (SCG) neurons from embryonic rats will be cultured in a two-chamber "Campenot" system and infected with fluorescently-labeled PRV. The chamber system enables the isolation of SCG cell bodies from their axon terminals and the neurons to which they connect, thereby separating the site of infection from the site of viral egress. We have found that spread from one chamber compartment to another requires intact axons; thus, in vitro viral spread is axon-mediated, as in vivo spread. PRV gD is an essential viral ligand required for the entry and fusion of extracellular virions to cells by binding various cellular receptors including herpesvirus entry mediator (HVEM), nectin-1 and nectin-2. Using this two-chamber system, we have demonstrated that gD is not required for trans-neuronal spread, implying that mature extracellular infectious particles may not be involved in this neuron-to-neuron spread. We also have shown that PRV-Bartha, an attenuated virus defective in anterograde spread, fails to spread from one chamber to the next, thus faithfully recapitulating in vitro what has been observed in vivo neural circuits. These chambers will be used to define both PRV and host genes required for trans-neuronal spread. In addition, live imaging of individual viral particles spreading to target cells should illuminate how the virus enters the target neurons, and more specifically, if the infection spreads via synaptic connections.

In vivo methods: The submandibular ganglion (SMG) has been used for imaging of synapses during development and will serve as an in vivo model for viral spread. Injection of fluorescently labeled PRV strains into surgically exposed salivary glands in anesthetized mice results specifically in the infection of the SMG, and not the surrounding tissues. Upon spread from the salivary tissue to the SMG, PRV undergoes replication in SMG cell bodies and subsequently spreads to the superior salivatory nucleus (SSN) located in the brain stem. We have focused our studies on the retrograde movements of individual viral particles traveling from the submandibular ganglion cells to the SSN axon termini. To aid in the visualization of individual SSN-SMG synapses by two-photon microscopy, we have utilized transgenic mice that express thy1-YFP specifically in the SSN cell bodies, axons and synaptic boutons. SMG have small post-synaptic “spikes” rather than dendrites, subsequently, the YFP+ boutons synapse close to the cell bodies. We have simultaneously imaged individual viral particles and synaptic boutons by TPLSM using Thy1-YFP mice infected with PRV expressing a CFP-capsid fusion protein. SBFSEM allows for the semi-automated acquisition of 3D datasets at nanoscopic resolution. In collaboration with Winfried Denk’s lab (MPI, Heidelberg, Germany), we have used SBFSEM to collect datasets of viral distribution and organization within an infected ganglion cells. Using this technique, we have found “pockets” of virus in the post-synaptic “spikes” and in the pre-synaptic axons emanating from the brain stem, suggesting that PRV uses synapses for spread between neurons.

FINDINGS

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General Summary of Findings: Several neuroinvasive viruses can be used to study the mammalian nervous system. In particular, infection by pseudorabies virus (PRV), an a-herpesvirus with broad host range, reveals chains of functionally-connected neurons in the nervous systems of a variety of mammals. The specificity of PRV trans-neuronal spread has been established in several systems using rather standard methods of histology and microscopy. The work supported by this grant enabled us to develop and apply novel imaging approaches, enabling us to study infection in living cells and tissues. Modern imaging technology will provide a more detailed understanding of the architecture of the nervous system as well as insights into processes of neuronal infection.

SELECTED PUBLICATIONS

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Feierbach B., Bisher M., Goodhouse J., and Enquist L.W. In vitro analysis of transneuronal spread of an alphaherpesvirus infection in peripheral nervous system neurons. J Virol. 2007 Jul;81(13):6846-57.

Smith G.A., Pomeranz L., Gross S.P., and Enquist L.W.. Local modulation of plus-end transport targets herpesvirus entry and egress in sensory axons. Proc Natl Acad Sci U S A. 2004 Nov 9;101(45):16034-9.

Enquist L.W. Exploiting circuit-specific spread of pseudorabies virus in the central nervous system: insights to pathogenesis and circuit tracers. J Infect Dis. 2002 Dec 1;186 Suppl 2:S209-14.

Lyman MG, Feierbach B, Curanovic D, Bisher M, Enquist LW. Pseudorabies virus us9 directs axonal sorting of viral capsids. J Virol., 81(20):11363-11371, 2007.

Ch'ng TH, Spear PG, Struyf F, Enquist LW. Glycoprotein D-independent spread of pseudorabies virus infection in cultured peripheral nervous system neurons in a compartmented system. J Virol., 81(19):10742-10757, 2007.

Viney TJ, Balint K, Hillier D, Siegert S, Boldogkoi Z, Enquist LW, Meister M, Cepko CL, Roska B. Local Retinal Circuits of Melanopsin- Containing Ganglion Cells Identified by Transsynaptic Viral Tracing. Curr Biol., 17(11):981-988, 2007.

Favoreel HW, Enquist LW, Feierbach B. Actin and Rho GTPases in herpesvirus biology. Tr Microbiol., 15(9):426-433, 2007.

Feierbach B, Piccinotti S, Bisher M, Denk W, Enquist LW. Alpha-herpesvirus infection induces the formation of nuclear actin filaments. PLoS Pathog., 2(8):e85, 2006.