Herpes Simplex Virus Type 1 Glycoprotein E Mediates Retrograde Spread from Epithelial Cells to Neurites

In animal models of infection, glycoprotein E (gE) is required for efficient herpes simplex virus type 1 (HSV-1) spread from the inoculation site to the cell bodies of innervating neurons (retrograde direction). Retrograde spread in vivo is a multistep process, in that HSV-1 first spreads between epithelial cells at the inoculation site, then infects neurites, and finally travels by retrograde axonal transport to the neuron cell body. To better understand the role of gE in retrograde spread, we used a compartmentalized neuron culture system, in which neurons were infected in the presence or absence of epithelial cells. We found that gE-deleted HSV-1 (NS-gEnull) retained retrograde axonal transport activity when added directly to neurites, in contrast to the retrograde spread defect of this virus in animals. To better mimic the in vivo milieu, we overlaid neurites with epithelial cells prior to infection. In this modified system, virus infects epithelial cells and then spreads to neurites, revealing a 100-fold retrograde spread defect for NS-gEnull. We measured the retrograde spread defect of NS-gEnull from a variety of epithelial cell lines and found that the magnitude of the spread defect from epithelial cells to neurons correlated with epithelial cell plaque size defect, indicating that gE plays a similar role in both types of spread. Therefore, gE-mediated spread between epithelial cells and neurites likely explains the retrograde spread defect of gE-deleted HSV-1 in vivo.Herpes simplex virus type 1 (HSV-1) is an alphaherpesvirus that characteristically infects skin and mucosal surfaces before spreading to sensory neurons, where it establishes a lifelong persistent infection. The virus periodically returns to the periphery via sensory axons and causes recurrent lesions as well as asymptomatic shedding. This life cycle requires viral transport along axons in two directions: toward the neuron cell body (retrograde direction) and away from the neuron cell body (anterograde direction).Many studies of alphaherpesvirus neuronal spread have focused on pseudorabies virus (PRV), a virus whose natural host is the pig. Three PRV proteins, glycoprotein E (gE), gI, and Us9, have been shown to mediate anterograde neuronal spread both in animal models of infection and in cultured neurons. However, these three proteins are dispensable for retrograde spread (3, 8, 11, 12, 31, 46). In contrast, numerous animal models of infection have shown that HSV-1 gE is required for retrograde spread from the inoculation site to the cell bodies of innervating neurons (4, 9, 44, 56). In the murine flank model, wild-type (WT) virus replicates in the skin and then infects sensory neurons and spreads in a retrograde direction to the dorsal root ganglia (DRG). In this model, gE-deleted HSV-1 replicates in the skin but is not detected in the DRG (9, 44). This phenotype differs from gE-deleted PRV, which is able to reach the DRG at WT levels (8). Thus, unlike PRV, gE-deleted HSV-1 viruses have a retrograde spread defect in vivo.HSV-1 gE is a 552-amino-acid type I membrane protein found in the virion membrane as well as in the trans-Golgi and plasma membranes of infected cells (1). gE forms a heterodimer with another viral glycoprotein, gI. The gE/gI complex is important for HSV-1 immune evasion through its Fc receptor activity. gE/gI binds to the Fc domain of antibodies directed against other viral proteins, sequestering these antibodies and blocking antibody effector functions (27, 32, 40). Additionally, gE/gI promotes spread between epithelial cells. Viruses lacking either gE or gI form characteristically small plaques in cell culture and small inoculation site lesions in mice (4, 9, 18, 40, 58). In animal models, gE and gI also mediate viral spread in both anterograde and retrograde directions (4, 19, 44, 56).In order to better understand the role of gE in HSV-1 retrograde neuronal spread, we employed a compartmentalized neuron culture system that has been used to study directional neuronal spread of PRV and West Nile virus (12, 14, 45). In the Campenot chamber system, neurites are contained in a compartment that is separate from their corresponding cell bodies. Therefore, spread in an exclusively retrograde direction can be measured by infecting neurites and detecting spread to neuron cell bodies.HSV-1 replication requires retrograde transport of incoming viral genomes to the nucleus. In neurites, fusion between viral and cellular membranes occurs at the plasma membrane (43, 48). Upon membrane fusion, the capsid and a subset of tegument proteins (the inner tegument) dissociate from glycoproteins and outer tegument proteins, which remain at the plasma membrane (28, 38). Unenveloped capsids and the associated inner tegument proteins are then transported in the retrograde direction to the nucleus (7, 48, 49).For both neurons and epithelial cells, retrograde transport is dependent upon microtubules, ATP, the retrograde microtubule motor dynein, and the dynein cofactor dynactin (22, 34, 49, 52). Several viral proteins interact with components of the dynein motor complex (23, 39, 60). However, none of these proteins suggest a completely satisfactory mechanism by which viral retrograde transport occurs, either because they are not components of the complex that is transported to the nucleus (UL34, UL9, VP11/12) or because capsids lacking that protein retain retrograde transport activity (VP26) (2, 17, 21, 28, 37). This implies that additional viral proteins are involved in retrograde trafficking.We sought to better characterize the role of gE in retrograde spread and found that gE is dispensable for retrograde axonal transport; however, it promotes HSV-1 spread from epithelial cells to neurites. This epithelial cell-to-neuron spread defect provides a plausible explanation for the retrograde spread defect of gE-deleted HSV-1 in animal models of infection.