Herpes Simplex Virus with Highly Reduced gD Levels Can Efficiently Enter and Spread between Human Keratinocytes

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The herpes simplex virus (HSV) glycoprotein heterodimer gE/gI plays an important role in virus cell-to-cell spread in epithelial and neuronal tissues. In an analogous fashion, gE/gI promotes virus spread between certain cell types in culture, e.g., keratinocytes and epithelial cells, cells that are polarized or that form extensive cell junctions. One mechanism by which gE/gI facilitates cell-to-cell spread involves selective sorting of nascent virions to cell junctions, a process that requires the cytoplasmic domain of gE. However, the large extracellular domains of gE/gI also appear to be involved in cell-to-cell spread. Here, we show that coexpression of a truncated form of gE and gI in a human keratinocyte line, HaCaT cells, decreased the spread of HSV between cells. This truncated gE/gI was found extensively at cell junctions. Expression of wild-type gE/gI that accumulates at intracellular sites, in the trans-Golgi network, did not reduce cell-to-cell spread. There was no obvious reduction in production of infectious HSV in cells expressing gE/gI, and virus particles accumulated at cell junctions, not at intracellular sites. Expression of HSV gD, which is known to bind virus receptors, also blocked cell-to-cell spread. Therefore, like gD, gE/gI appears to be able to interact with cellular components of cell junctions, gE/gI receptors which can promote HSV cell-to-cell spread.The alphaherpesviruses herpes simplex virus (HSV) types 1 (HSV-1) and 2, varicella-zoster virus (VZV), and pseudorabies virus (PRV) replicate primarily in epithelial tissues before spreading into neurons where they establish latency. These viruses are extraordinarily adept at spreading rapidly from cell to cell in both these tissues. Given that alphaherpesviruses establish lifelong infections, cell-to-cell spread appears to be especially important in order for these viruses to outrun the immune system, especially after reactivation in hosts that have fully primed immunity. Consistent with this, HSV, PRV, and VZV all remain largely cell associated; large numbers of progeny virions accumulate on cell surfaces, especially at cell junctions in polarized epithelial cells (23). Moreover, virus mutants with specific defects in cell-to-cell spread that do not affect entry at apical surfaces are extremely attenuated in vivo (reviewed in references 15 and 21). It has been proposed elsewhere that alphaherpesviruses have evolved specialized mechanisms to allow specific sorting of virions to epithelial and neuronal cell junctions and the efficient transfer across junctions to promote infection of adjacent cells (14,21).HSV, PRV, and VZV all express a glycoprotein heterodimer, gE/gI, that promotes cell-to-cell spread (19,20,25,49,50). In the case of HSV and PRV, gE/gI was originally thought to be nonessential based on observations that gE Ϫ or gI Ϫ mutants enter cultured laboratory cells and replicate normally in the cells. However, PRV and HSV gE Ϫ or gI Ϫ mutants are severely compromised in epithelial and neuronal tissues (1,6,7,12,13,33,37,43,44,46...

Section: Discussionmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Herpes Simplex Virus gE/gI Expressed in Epithelial Cells Interferes with Cell-to-Cell Spread

Collins

Johnson

2003

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“…The viral glycoproteins are expressed at high levels in the fusion assay, and we have not tested the effect of reduced expression levels or of changes in their relative amounts. We do not know how many gD-receptor interactions are required per virion to initiate entry, but the HSV mutant FUS6kan, which contains 500-fold less gD than does WT virus, is viable only on cells that express large amounts of receptor (21).…”

Section: Discussionmentioning

confidence: 99%

Function of Herpes Simplex Virus Type 1 gD Mutants with Different Receptor-Binding Affinities in Virus Entry and Fusion

Milne

Hanna

Rux

et al. 2003

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We have studied the receptor-specific function of four linker-insertion mutants of herpes simplex virus type 1 glycoprotein D (gD) representing each of the functional regions of gD. We used biosensor analysis to measure binding of the gD mutants to the receptors HVEM (HveA) and nectin-1 (HveC). One of the mutants, gD(١34t), failed to bind HVEMt but showed essentially wild-type (WT) affinity for nectin-1t. The receptor-binding kinetics and affinities of the other three gD mutants varied over a 1,000-fold range, but each mutant had the same affinity for both receptors. All of the mutants were functionally impaired in virus entry and cell fusion, and the levels of activity were strikingly similar in these two assays. gD(١34)-containing virus was defective on HVEM-expressing cells but did enter nectin-1-expressing cells to about 60% of WT levels. This showed that the defect of this form of gD on HVEM-expressing cells was primarily one of binding and that this was separable from its later function in virus entry. gD(١243t) showed WT binding affinity for both receptors, but virus containing this form of gD had a markedly reduced rate of entry, suggesting that gD(١243) is impaired in a postbinding step in the entry process. There was no correlation between gD mutant activity in fusion or virus entry and receptor-binding affinity. We conclude that gD functions in virus entry and cell fusion regardless of its receptor-binding kinetics and that as long as binding to a functional receptor occurs, entry will progress.Entry of herpes simplex virus type 1 (HSV-1) into cells involves the coordinated actions of at least five viral glycoproteins: glycoprotein B (gB), gC, gD, gH, and gL (50). All but gC are indispensable for entry (1,13,27,43), and gB, gD, gH, and gL are sufficient for cell fusion (55). With the exception of gD, the individual functions served by the essential glycoproteins are unknown. gD is a type I virion membrane glycoprotein of 369 amino acids in its mature form. The core of gD (residues 56 to 184) has a V-like immunoglobulin (Ig) fold. This is encircled by extensions at the N and C termini which comprise the principal functional domains of the protein (3). gD homologues are present only in the alphaherpesviruses (although not in varicella-zoster virus). In these viruses, gD plays a conserved and pivotal role during virus entry, binding to one of several specific receptors (2,5,17,36,51,56). Two of these receptors, herpesvirus entry mediator (HVEM; also known as HveA) and nectin-1 (CD111; previously known as HveC), have been extensively studied because they are used by all wild-type (WT) strains of HSV-1 (17, 36).HVEM is a member of the tumor necrosis factor receptor family (36). It has at least two natural ligands, LIGHT and lymphotoxin-␣, as well as gD (31,47). Nectin-1 is a cell surface protein comprising one V-like and two C-like Ig domains (28). The N-terminal V domain contains the gD-binding region (23,30,32). Nectin-1 functions as an adhesion molecule and is found at adherens junctions, where it can e...

“…The cell-to-cell spread defects of HSV-1 gE mutants have been reported mainly for polarized cell types (25,30,41,59). In an electron microscopy study, WT virions localized to the basolateral surface of polarized HEC-1-A and HaCaT cells, whereas gE-deleted virions localized to the apical surface (24,33).…”

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confidence: 99%

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

McGraw

Friedman

2009

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-...