Membrane Requirement for Folding of the Herpes Simplex Virus 1 gB Cytodomain Suggests a Unique Mechanism of Fusion Regulation

Abstract

bHerpes simplex virus type 1 (HSV-1) enters cells by fusion of its envelope with a host cell membrane, which requires four viral glycoproteins and a cellular receptor. Viral fusion glycoprotein B (gB) mediates membrane fusion through the action of its ectodomain, while its cytoplasmic domain (cytodomain) regulates fusion from the opposite face of the membrane by an unknown mechanism. The gB cytodomain appears to restrict fusion, because point or truncation mutations within it increase the extent of fusion (syn mutations). Previously, we showed that the hyperfusion phenotype correlated with reduced membrane binding in gB syn truncation mutants and proposed that membrane binding was important in regulating fusion. Here, we extended our analysis to three syn point mutants: A855V, R858H, and A874P. These mutations produce local conformational changes, with some affecting membrane interaction, which suggests that while syn mutants may deregulate fusion by somewhat different mechanisms, maintaining the wild-type (WT) conformation is critical for fusion regulation. We further show that the presence of a membrane is necessary for the cytodomain to achieve its fully folded conformation and propose that the membrane-bound form of the cytodomain represents its native conformation. Taken together, our data suggest that the cytodomain of gB regulates fusion by a novel mechanism in which membrane interaction plays a key role. Herpes simplex virus type 1 (HSV-1) causes diseases ranging from mild skin lesions to fatal encephalitis and neonatal herpes (28). Like all herpesviruses, HSV-1 enters host cells by fusion of the viral envelope with the plasma membrane or an endocytic vesicle. While the specific viral glycoproteins required for entry differ among herpesviruses, glycoprotein B (gB) and the heterodimer gH/gL are conserved. Entry of HSV-1 requires gD, gB, and gH/gL, in addition to a host cell receptor (Fig. 1A). These five proteins are also sufficient to mediate fusion of transiently transfected cells in the absence of any other viral proteins (47).gD initiates entry by binding one of its three cellular receptors (44). The exact role of gH/gL in entry remains unclear; however, recent evidence suggests that binding of a receptor by gD activates gH/gL, which in turn activates gB for fusion (3). gB is a class III viral fusion protein that enables membrane fusion, presumably by undergoing dramatic conformational changes that provide the energy to drive fusion (25, 29). However, unlike fusion proteins of other enveloped viruses, e.g., influenza virus hemagglutinin (HA) or HIV gp160, which accomplish fusion independently (15), HSV-1 gB mediates fusion only in the presence of gD, a gD receptor, and gH/gL (39, 47). gB is a trimeric, type I transmembrane protein with an N-terminal ectodomain displayed on the virion surface, a single-pass transmembrane helix, and a C-terminal cytoplasmic domain (cytodomain) situated on the opposite face of the viral envelope (29) (Fig. 1A). The ectodomain is presumably directly involved in the membra...

An immunoreceptor tyrosine-based inhibition motif in varicella-zoster virus glycoprotein B regulates cell fusion and skin pathogenesis

“…The revised conformation of two independent α-helices on either side of the ITIM would leave the tyrosine prominently exposed to serve as a substrate for kinases similar to the VZV gBcyt. It has been proposed that the HSV gBcyt membrane association in its native conformation restricts fusogenic activity (20). Combined with our findings, these studies strongly support the conclusion that the ITIM is a critical factor in gBcytdependent fusogenicity.…”

“…Therefore, gBcyt structure was unlikely to have deformed sufficiently to generate the exaggerated cell-fusion phenotypes associated with these phenylalanine substitutions. Conversely, structural changes could result from gBcyt truncations or natural or engineered mutations described in previous herpesvirus studies, altering the gBcyt conformation or hydrophobicity and, subsequently, its attributes for membrane association (20,23,24). Although effects on the gB ectodomain structure cannot be discounted, studies of HSV-2 gB based on reactivity with monoclonal antibodies indicate that the ectodomain is not distorted in gBcyt mutants with syncytial phenotypes (20).…”

“…Mutations in this hot spot could deform the HSV gBcyt to inhibit tyrosine phosphorylation, resulting in a "syn" phenotype similar to the exaggerated syncytia caused by the Y881F substitution in the gBcyt ITIM. Recent investigations show that the HSV-2 gBcyt forms a protease-resistant core situated around this conserved ITIM in the presence of lipid membranes (20). The revised conformation of two independent α-helices on either side of the ITIM would leave the tyrosine prominently exposed to serve as a substrate for kinases similar to the VZV gBcyt.…”

“…These tyrosines were replaced with phenylalanine to create three mutant gB constructs, Y881F, Y920F, and Y881/920F (Table S2). Phenylalanine rather than alanine substitution was chosen to retain the gBcyt structure, which has been reported to affect HSV-1 gB function (20). These mutants were compared with gB constructs with an L884G mutation that inhibits endocytosis and with two gB ectodomain mutants, W180G and Y185G (Fig.…”

“…Whereas syncytia formation is typical of VZV, engineered or spontaneous substitutions in the gBcyt in other herpesviruses lead to syncytial herpesvirus phenotypes (15)(16)(17). The cytoplasmic domains of gB homologs contain α-helices and endocytosis motifs (18)(19)(20). Endocytosis, linked to dileucine and YXXΦ motifs (X, any amino acid; Φ, a large hydrophobic residue), is necessary for gB cellsurface retrieval, trans-Golgi network (TGN) localization, and incorporation into the virion envelope (21).…”