Random linker-insertion mutagenesis to identify functional domains of herpes simplex virus type 1 glycoprotein B

Abstract

Herpes simplex virus glycoprotein B (gB) is one of four glycoproteins essential for viral entry and cell fusion. Recently, an x-ray structure of the nearly full-length trimeric gB ectodomain was determined. Five structural domains and two linker regions were identified in what is probably a postfusion conformation. To identify functional domains of gB, we performed random linkerinsertion mutagenesis. Analyses of 81 mutants revealed that only 27 could fold to permit processing and transport of gB to the cell surface. These 27 mutants fell into three categories. Insertions into two regions excluded from the solved structure (the N terminus and the C-terminal cytoplasmic tail) had no negative effect on cell fusion and viral entry activity, identifying regions that can tolerate altered structure without loss of function. Insertions into a disordered region in domain II and the adjacent linker region also permitted partial cell fusion and viral entry activity. Insertions at 16 other positions resulted in loss of cell fusion and viral entry activity, despite detectable levels of cell surface expression. Four of these insertion sites were not included in the solved structure. Two were between residues exposed to a cavity that is too small to accommodate the 5-amino acid insertions, consistent with the solved structure being different from the native prefusion structure. Ten were between residues exposed to the surface of the trimer, identifying regions that may be critical for interactions with other viral proteins or cellular components or for transitions from the prefusion to postfusion state.cell fusion ͉ glycoprotein B ͉ viral entry ͉ mutation H erpes simplex virus (HSV) is a neurotropic virus that can cause recurrent mucocutaneous lesions of the oral or genital epithlelium, lesions on the cornea and, rarely, encephalitis. Infection of host cells occurs through virus attachment to the cell surface and subsequent membrane fusion to deliver the nucleocapsid containing the viral genome into the host cell. Virus attachment is mediated by binding of glycoproteins C (gC) or B (gB) to cell surface glycosaminoglycans, primarily heparan sulfate (1). Subsequent fusion between the virion envelope and a host cell membrane requires gB, glycoprotein D (gD), and the heterodimer glycoprotein H (gH)-glycoprotein L (gL) and one of the cellular receptors for gD. These receptors include herpesvirus entry mediator (HVEM), a member of the TNF receptor family; nectin-1 and nectin-2, cell adhesion molecules of the Ig superfamily; and specific sites in heparan sulfate generated by 3-O-sulfotransferases (2). It has been proposed that binding of gD to one of its cellular receptors induces gD to undergo a conformational change, resulting in interactions with gB and/or the gH-gL complex to trigger membrane fusion (3, 4).The exact roles for gB and gH-gL in the membrane fusion process have yet to be elucidated. Recently, it was shown (5) that gH-gL, along with gD and a gD receptor, were sufficient to induce hemifusion, mixing of the outer leafle...

Insertion Mutations in Herpes Simplex Virus 1 Glycoprotein H Reduce Cell Surface Expression, Slow the Rate of Cell Fusion, or Abrogate Functions in Cell Fusion and Viral Entry

“…A previous study also reported a panel of gH insertional mutants in which between 57 and 76% of mutants were expressed normally, depending on the antibody used to detect expression (11). In similar studies done with gD (7) and gB (26), cell surface expression was achieved for 86% and 33% of mutants, respectively. This difference between the panels of gH and gB insertion mutants generated in an identical fashion may indicate that gH, unlike gB, has a more flexible conformation and performs functions that are not so dependent on intrachain interfaces or dramatic conformational changes.…”

“…Target cells were transfected with 400 ng of plasmid containing luciferase and 1.8 g of empty vector. After 4 h of transfection, cells were washed, detached, and overlaid as described elsewhere previously (26). After coincubation for the indicated times, cells were washed and lysed, and luciferase activity was measured as described above.…”

Herpes Simplex Virus 1 Glycoprotein B and US3 Collaborate To Inhibit CD1d Antigen Presentation and NKT Cell Function

“…gB is the most conserved glycoprotein in the Herpesviridae family and plays a key role in mediating fusion between viral envelope and host cell membrane during viral entry (65). Due to its fusogenic function, its cell surface expression must be tightly regulated (42). Recently, the viral serine/threonine kinase US3 has been found to play a key role in modulating gB cell surface expression (31,40,72), suggesting that it might collaborate with gB in affecting CD1d expression.…”

Insertional Mutations in Herpes Simplex Virus Type 1 gL Identify Functional Domains for Association with gH and for Membrane Fusion

“…CHO cells seeded in 96-well plates were transfected with 20 ng of plasmid expressing WT gH or empty vector, a plasmid expressing WT gL or a gL mutant, and 0.13 l of Lipofectamine 2000 diluted in Opti-MEM (Gibco). The cells were washed once with phosphatebuffered saline (PBS) 24 h after transfection, and a cell-based enzyme-linked immunosorbent assay (CELISA) was performed as described previously (19). Briefly, after incubation of the transfected cells with the antibodies, the cells were washed, fixed, and incubated with biotinylated goat anti-rabbit immunoglobulin G or biotinylated rabbit anti-mouse immunoglobulin G (Sigma), followed by streptavidin-horseradish peroxidase (GE Healthcare) and horseradish peroxidase substrate (BioFX).…”