Pseudorabies Virus Membrane Proteins gI and gE Facilitate Anterograde Spread of Infection in Projection- Specific Neurons in the Rat

Alphaherpesviruses, like Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), Varicella-zoster virus, and Pseudorabies virus (PRV), exhibit a predilection to infect neurons in the central nervous system (CNS) and peripheral nervous system (PNS) of an animal host (9a). During natural infections, virus replicates in nonneuronal cells and then undergoes retrograde spread of virus into afferent (e.g., sensory) or efferent (e.g., motor) nerve fibers innervating the infected tissue. Typically, this viral infection is limited to the PNS, where a latent infection is established in neurons directly innervating the peripheral site. Reactivation from latency is associated with virus replication in the primary neuron followed by anterograde transport of newly synthesized virus particles from the neuronal cell body back to the innervated tissue. Although less frequent, virus may also spread from the PNS to the CNS following primary infection or reactivation; this often has fatal consequences for the host. Hence, alphaherpesviruses are likely to encode mechanisms that facilitate travel in either direction within a neural circuit and thus may regulate the choice of direction at different stages of the virus life cycle (9a). Insight that glycoproteins gE and gI are potential viral gene products involved in these mechanisms comes from studies demonstrating reduced infection of CNS after peripheral infection. For example, after intravitreal inoculation of the rat eye with wild-type PRV, virus replicates in retinal ganglion neurons and then undergoes anterograde spread to all visual nuclei in the CNS (6, 7, 31). By contrast, gI and gE mutant viruses replicate well in the eye but do not spread to second-order CNS neurons in a major subset of visual centers such as the superior colliculus and the dorsal lateral geniculate nucleus (dLGN). A similar inability to infect a subset of cardiac neurons in the rat brain stem has been reported following inoculation of gE mutants into the heart (26, 27). Similar defects in directional spread of infection have been noted after infection of mice by PRV gE and gI mutants (1). Importantly, similar defects have been seen after nasal infection by PRV gE and gI mutants of swine, the natural host (11, 13, 14, 18, 19). Like PRV, gE and gI deletion mutants of HSV-1 showed reduced neuroinvasion of the CNS in mice from peripherally infected sites (2, 21). While PRV gI and gE are required for efficient anterograde spread through most neuronal circuits tested, they are not required for retrograde spread (4, 5, 9a). Enquist et al. (9) suggested that failure of gI- and gE-negative viruses to produce a productive infection in second-order neurons in the rat visual system was not due to an inability of these mutants to enter and replicate in the first-order retinal ganglion cells. They demonstrated that coinfection of the retina with separate gE- and gI-defective strains resulted in a phenotypically wild-type infection with virus replication in all visual nuclei. This result suggested that the mutants complemented each other, which could occur only after initial cellular entry (9). Thus, the defect in anterograde spread must occur in the primary infected neuron. One or more steps of virus replication and spread may require the presence of gE and gI: (i) virus is produced in the neuronal cell body but transport into the axon does not occur; (ii) virus reaches the axon terminal but fails to exit; (iii) viral egress from the retinal ganglion cell axon is successful but virus fails to cross the synapse; (iv) virus receptor-mediated entry of the postsynaptic neuron fails; or (v) virus penetrates the second-order neuron, but this postsynaptic neuron is nonpermissive to mutant virus replication. The studies presented in this report were designed to determine the stage where gI- and gE-deficient viruses are blocked from infecting second-order superior colliculus neurons following intravitreal inoculation. First, to confirm and extend the conclusions of the coinfection experiments discussed above (9), we determined directly whether gI-gE double null mutant viruses were capable of infecting retinal ganglion cells that send axons to neurons in the superior colliculus. Second, we determined if gE and gI mutant viruses could replicate in superior colliculus neurons. Finally, we tested whether retinal ganglion neurons that project to the superior colliculus were competent to replicate gE and gI mutants by direct CNS injection and retrograde transport of virus back to the retina. (Portions of this work were taken from the undergraduate Senior Thesis of T.K.)