title: Structure-based mutational analysis of the highly conserved domain IV of glycoprotein H of pseudorabies virus.
abstract: Glycoprotein H (gH) is an envelope protein conserved in the Herpesviridae. Together with glycoprotein B (gB), the heterodimeric complex of gH and glycoprotein L (gL) mediates penetration and direct viral cell-to-cell spread. In herpes simplex and pseudorabies virus (PrV), coexpression of gH/gL, gB, and gD induces membrane fusion to form polykaryocytes. The recently determined crystal structure of a core fragment of PrV gH revealed marked structural similarity to other gH proteins (M. Backovic et al., Proc. Natl. Acad. Sci. U. S. A. 107:22635-22640, 2010). Within the membrane-proximal part (domain IV), a conserved negatively charged surface loop (flap) is flanked by intramolecular disulfide bonds. Together with an N-linked carbohydrate moiety, this flap covers an underlying patch of hydrophobic residues. To investigate the functional relevance of these structures, nonconservative amino acid substitutions were introduced by site-directed mutagenesis. The mutated proteins were tested for correct expression, fusion activity, and functional complementation of gH-deleted PrV. Several single amino acid changes within the flap and the hydrophobic patch were tolerated, and deletion of the glycosylation site had only minor effects. However, multiple alanine substitutions within the flap or the hydrophobic patch led to significant defects. gH function was also severely affected by disruption of the disulfide bond at the C terminus of the flap and after introduction of cysteine pairs designed to bridge the central part of the flap with the hydrophobic patch. Interestingly, all mutated gH proteins were able to complement gH-deleted PrV, but fusion-deficient gH mutants resulted in a pronounced delay in virus entry.
authors.affiliation: Institute of Molecular Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
authors.forename: Walter
authors.initials: W
authors.lastname: Fuchs
authors.forename: Marija
authors.initials: M
authors.lastname: Backovic
authors.forename: Barbara G
authors.initials: BG
authors.lastname: Klupp
authors.forename: Felix A
authors.initials: FA
authors.lastname: Rey
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Mettenleiter
mesh_terms: D000818:Animals; D002460:Cell Line; D011558:Herpesvirus 1, Suid / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008561:Membrane Fusion; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011557:Pseudorabies / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D014759:Viral Envelope Proteins