Mutations in the Cytoplasmic Tail of Herpes Simplex Virus 1 gH Reduce the Fusogenicity of gB in Transfected Cells

Abstract

Mutations within the cytoplasmic tail (cytotail) of herpes simplex virus 1 (HSV-1) gH were previously observed to suppress the syncytial phenotype of gB cytoplasmic domain mutant A855V in infected cells. Here, we examined the effects of gH cytotail mutations on virus-free cell-cell fusion in transfected cells to exclude the contributions of viral proteins other than gD, gH/gL, and gB. We show that a truncation at residue 832 coupled with the point mutation V831A within the cytotail of gH reduces fusion regardless of whether the wild type (WT) or a syn gB allele is present. We hypothesize that the gH cytotail mutations either reduce activation of gB by gH/gL or suppress the fusogenicity of gB through another, as yet unknown mechanism. The gB cytodomain and the gH cytotail do not interact in vitro, suggesting that mutations in the gH cytotail may instead affect the function of the gH/gL ectodomain. Nevertheless, we cannot exclude the possibility that the gB cytodomain and the gH cytotail interact in the context of full-length membrane-anchored proteins. The observed fusion suppression in transfected cells is less prominent than what was seen in infected cells, and we propose that gH cytotail mutations may additionally suppress syncytium formation in cells infected with syn HSV-1 by acting on other viral proteins, reinforcing the idea that fusion of HSV-infected cells is a complex phenomenon. Although fusion suppression by the gH cytotail mutant in transfected cells was evident when syncytia were visualized and counted, it was not detected by the luciferase assay, highlighting the differences between the two assays. Herpes simplex virus 1 (HSV-1) and HSV-2 are enveloped, double-stranded DNA viruses that cause mucocutaneous lesions, keratitis, and encephalitis (1). HSV enters host cells by fusing its envelope with the host cell membrane, which requires four viral envelope glycoproteins: glycoprotein D (gD), gB, gH, and gL (2-4) (Fig. 1A). In conjunction with a cellular receptor, these proteins also mediate fusion of transfected cells in the absence of other viral proteins (5, 6). Membrane fusion involving these and other glycoproteins also takes place during HSV spread from infected to uninfected cells (7,8).Fusion is initiated by binding of gD to one of its cellular receptors (9), which triggers a conformational change in gD (9, 10). This conformational change presumably activates the gH/gL heterodimer, which then likely activates gB (11). gB is a class III viral fusogen believed to catalyze membrane fusion by undergoing a conformational change from a prefusion to a postfusion form (12,13). gH/gL is thought to regulate the fusogenic potential of gB, likely through direct interaction of their ectodomains (11,14,15). Consistent with its regulatory role, gH/gL can function in trans (11, 16) and as a soluble complex (11), albeit with reduced efficiency.The HSV fusion machinery is subject to a multilevel regulatory mechanism. In addition to activation by gD-receptor interaction and by gH/gL, the fusion activity of gB ap...

Interplay between the Herpes Simplex Virus 1 gB Cytodomain and the gH Cytotail during Cell-Cell Fusion

“…7D), consistent with data from previous reports (33,36). The rest of the truncations progressively reduced the fusion activity of both WT gB and the hyperfusogenic mutants ( Fig.…”

“…Indeed, truncations resulted in a gradual reduction in fusion levels rather than an abrupt loss of activity at a specific length. Although no interaction between the recombinant gB cytodomain expressed in E. coli and a synthetic peptide encompassing the gH cytotail was detected in in vitro pulldown experiments (36), the interaction may be transient, or it may require the transmembrane regions (TMs) of gB and gH. Although the roles of the TMs in fusion have not yet been delineated, mutations in either TM reduce fusion (33,51).…”

“…The gH truncation gH832 was generated as described previously (36). The gH truncation gH828 was generated by SOE PCR using the primer pair 5=-AGCCTTCTGATAGCCTCGGCCCT GTGTACGT-3 and 5=-CGGGACTTATTACCGGAGAACCTTTAGGA-3= and the primer pair 5=-TCCTAAAGGTTCTCCGGTAATAAGTCCCGT TTTTTTG-3= and 5=-GTCCCCATAATTTTTGGCAGAGGGAAAAA-3= (double stop codons are in boldface type).…”

“…Additionally, truncations within the gH cytotail could suppress syncytium formation in cells infected with a syn HSV-1 strain (37) and showed delayed virus penetration (34). Previously, we showed that a gHSAP mutant, truncated at residue 832 and containing the point mutation V831A, reduced cell-cell fusion regardless of whether WT gB or a syn allele containing the A855V mutation was present (36). These results established the importance of the gH cytotail in regulating fusion and suggested that it participates at a rate-limiting step, yet its contribution to fusion has not yet been systematically investigated.…”

“…Like the gB cytodomain, the 14-amino-acid cytoplasmic tail (cytotail) of gH is necessary for fusion but may have an activating, rather than an inhibitory, role because truncations, insertions, or point mutations reduce cell-cell fusion of infected or transfected cells mediated by wild-type (WT) gB (33)(34)(35)(36). Additionally, truncations within the gH cytotail could suppress syncytium formation in cells infected with a syn HSV-1 strain (37) and showed delayed virus penetration (34).…”