Random linker-insertion mutagenesis to identify functional domains of herpes simplex virus type 1 glycoprotein B.
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 linker-insertion 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.
Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, MC S213, 320 East Superior Street, Chicago, IL 60611, USA.
Erick
E
Lin
Patricia G
PG
Spear
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000502:physiology; D016254:Mutagenesis, Insertional; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000502:physiology
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus