Research

Pseudorabies Virus

Genetics of directional spread of virus in and between neurons

Tracing the hardwiring of the nervous system using virus

Probing the host response to herpesviruses using microarrays

Genetics of Directional Spread of Virus In and Between Neurons

Axonal Sorting of Pseudorabies Virus
During alpha-herpesvirus egress in neurons, the capsid/tegument complex is released into the cytosol where it undergoes a secondary envelopment step into vesicles derived from the trans-Golgi network (TGN). These vesicles, containing enveloped virus particles, are then transported to the plasma membrane for local release from the cell body. Viral components are also targeted to the axon for long distance transport and release at axon termini. The mechanisms involved in axonal sorting are a key interest of our laboratory. Until recently it has been unclear what is actually sorted into axons, namely whether PRV capsid/tegument complexes are sorted separately from viral membrane proteins, or whether both are transported together as a mature virus particle contained within a vesicle. Using Campenot chamber technology coupled with transmission electron microscopy (TEM), we have been able to further elucidate this process.

The trichamber neuronal culturing system allows for the physical separation of neuronal cell bodies (soma) from axon termini. Dissociated rat embryonic neurons are plated in the soma (S) chamber and allowed to mature for two weeks. During this period, axons are directed between a series of grooves across the methocellulose (M) chamber to the neurite (N) chamber. Cell bodies in the S chamber are then infected, and virus structural components are sorted into axons in the anterograde direction (into the middle and neurite compartments). The initial infection is confined to the S chamber via silicone vacuum grease and a methocellulose barrier. Therefore, input inoculum or newly replicated virus released from cell bodies is unable to confound analyses examining virus components sorted into the axon. In the experiment shown above, wild-type Becker was used to infect cell bodies in the soma compartment, and EM analysis was subsequently performed in the M compartment to examine the nature of viral capsids in axons. These findings suggested that mature virus particles, found within a transport vesicle, were directed into axons.

Our lab has also developed another in vitro chamber system that physically separates cell bodies from axon termini. Rat embryonic ganglion explants are plated and allowed to extend axons for one week in the presence of nerve growth factor (NGF). A non-septated, Teflon chamber disk is then placed on top of the axons thereby capturing a subpopulation of axon ends. One can then infect the explant and image inside the chamber ring to examine anterograde transport of viral structural proteins. EM analysis of axons from Becker infected explants also showed the presence of enveloped virus particles within a vesicle, supporting previous data generated in the trichamber system. The notion that mature virus particles are sorted into the axon has been further supported by live-cell imaging experiments using GFP and RFP tagged structural proteins (Greg Smith, Northwestern University) in addition to indirect immunofluorescene of infected neurons in the isolator chamber system (our lab). The idea that virus encodes a mechanism to direct trafficking of cellular vesicles into the axon (which may or may not contain a virus particle) is very intriguing. What viral genes are involved in this process?

PRV Us9 Is Primarily Responsible for Axonal Sorting of Virus Particles
The PRV Us9 gene product is a phosphorylated, type II membrane protein that is "tail-anchored," meaning that it has no identifiable signal sequence, and has an amino-terminus that resides in the cytosol, and a carboxy-terminal anchor that spans the lipid bilayer. Its steady state concentration is highest in or near the trans-Golgi network, and is heavily enriched in lipid raft microdomains. The absence of Us9 does not affect cell-to-cell spread in Madin-Darby bovine kidney cells, but has a dramatic effect on anterograde spread of infection in the visual circuitry of the rat brain and neuron-to-cell spread in vitro, suggesting that Us9 performs a neuronal specific function during replication. Work in our lab has shown that Us9 is essential for the anterograde transport of viral capsids as well as viral glycoproteins. The heterodimeric complex gE/gI also impacts the sorting of virus particles into the axon, but to a lesser extent than Us9. Thus, our model is that gE/gI may play an accessory role to Us9, and that all three proteins working together lead to efficient axonal sorting of vesicles (see below).

We propose a model in which Us9, gE/gI, and lipid rafts direct the sorting of vesicles into the axon of infected neurons. Us9 and gE/gI likely associate with lipid rafts in the trans-Golgi network (TGN), the proposed site of viral assembly. The presence of Us9 and gE/gI in lipid rafts (those decorating the surface of cellular vesicles) would recruit axonal sorting machinery to a small number of viral assembly complexes in the TGN, i.e. vesicles with viral membrane proteins only, those containing mature virus particles, or L-particles. A limited number of vesicles containing virion components would then be targeted to the axon.