Plasma Membrane Topology of Syncytial Domains of Herpes Simplex Virus Type 1 Glycoprotein K (gK): the UL20 Protein Enables Cell Surface Localization of gK but Not gK-Mediated Cell-to-Cell Fusion

Abstract

Most spontaneously occurring mutations that cause extensive herpes simplex virus type 1 (HSV-1)-induced cell fusion are single amino acid changes within glycoprotein K (gK). Despite the strong genetic association of gK with virus-induced cell fusion, its direct involvement in cellular membrane fusion has been controversial, largely due to previously unsuccessful efforts to detect gK expression on virion and cellular surfaces. Recently, we showed that gK is expressed on HSV-1 virions and functioned in virus entry (T. P. Foster, G. V. Rybachuk, and K. G. Kousoulas, J. Virol. 75:12431-12438, 2001). To determine whether gK is expressed on cellular surfaces, as well as its membrane topology, we generated the recombinant viruses gKV5DI, gKV5DII, gKV5DIII, and gKV5DIVcontaining insertions of the V5 antigenic epitope within each of four domains of gK predicted to localize either in the cytoplasmic side or in the extracytoplasmic side of cellular membranes. Immunohistochemical and confocal microscopy analyses of infected cells showed that both wild-type and syncytial forms of gK were expressed on cell surfaces. Analysis of the topology of the V5-tagged gK revealed that gK domains I and IV were located extracellularly, whereas domains II and III were localized intracellularly. Transiently expressed gK failed to localize in cellular plasma membranes. In contrast, infection of gKtransfected cells with the gK-null virus ⌬gK enabled expression of gK on cell surfaces, as well as gK-mediated membrane fusion. Transient-coexpression experiments revealed that the UL20 protein enabled cell surface expression of gK, but not gK-mediated cell-to-cell fusion, indicating that additional viral proteins are required for expression of the gK syncytial phenotype.Viral glycoproteins are key determinants of membrane fusion events throughout the life cycle of herpesviruses. Herpes simplex viruses (HSVs) specify at least 11 glycoproteins:

Differential importance of highly conserved residues in UL24 for herpes simplex virus 1 replication in vivo and reactivation

“…UL20 and gK are involved in secondary envelopment of virions (Baines et al, 1991;Foster & Kousoulas, 1999;Fuchs et al, 1997;Hutchinson & Johnson, 1995;Jayachandra et al, 1997). It has been shown that UL20 is required for the correct trafficking and localization of gK (Foster et al, 2003). In addition, a UL20-null mutation can suppress fusion in the context of infection with a virus carrying a syn mutation in gK or gB (Foster et al, 2004).…”

Contributions of Herpes Simplex Virus 1 Envelope Proteins to Entry by Endocytosis

“…Extracellular gK Ϫ and UL20 Ϫ HSVs were not obtained in sufficient quantities compared to the other viruses tested in this study. This is due to the critical roles of gK and UL20 in HSV egress (30,(40)(41)(42). However, cell-associated preparations of gK Ϫ and UL20…”

A Recombinant Herpes Simplex Virus Type 1 Expressing Two Additional Copies of gK Is More Pathogenic than Wild-Type Virus in Two Different Strains of Mice

“…However, our preliminary results have shown that these regulatory T cells are not detectable in the corneas of infected mice (data not shown). Previously, we have shown that the overexpression of gK in gK-transformed cells collapses the Golgi apparatus into the endoplasmic reticulum (10). Thus, the disruption of the Golgi structure by the overexpression of gK in the HSVgK 3 virus may also damage cellular structure of the eye that may result in the exacerbation of CS in infected mice.…”

“…This exacerbated CS and facial dermatitis was due to the presence of CD8 ϩ T cells in the cornea of ocularly infected mice (44). Furthermore, we have shown that the overexpression of gK in gK-transformed cells causes a collapse of the Golgi apparatus into the endoplasmic reticulum, which inhibits virion egress, glycoprotein transport, and virus-induced cell fusion (10).…”