Fusion-Deficient Insertion Mutants of Herpes Simplex Virus Type 1 Glycoprotein B Adopt the Trimeric Postfusion Conformation

Glycoprotein B (gB) enables the fusion of viral and cell membranes during entry of herpesviruses. However, gB alone is insufficient for membrane fusion; the gH/gL heterodimer is also required. The crystal structure of the herpes simplex virus type 1 (HSV-1) gB ectodomain, gB730, has demonstrated similarities between gB and other viral fusion proteins, leading to the hypothesis that gB is a fusogen, presumably directly involved in bringing the membranes together by refolding from its initial or prefusion form to its final or postfusion form. The only available crystal structure likely represents the postfusion form of gB; the prefusion form has not yet been determined. Previously, a panel of HSV-1 gB mutants was generated by using random 5-amino-acid-linker insertion mutagenesis. Several mutants were unable to mediate cell-cell fusion despite being expressed on the cell surface. Mapping of the insertion sites onto the crystal structure of gB730 suggested that several insertions might not be accommodated in the postfusion form. Thus, we hypothesized that some insertion mutants were nonfunctional due to being “trapped” in a prefusion form. Here, we generated five insertion mutants as soluble ectodomains and characterized them biochemically. We show that the ectodomains of all five mutants assume conformations similar to that of the wild-type gB730. Four mutants have biochemical properties and overall structures that are indistinguishable from those of the wild-type gB730. We conclude that these mutants undergo only minor local conformational changes to relieve the steric strain resulting from the presence of 5 extra amino acids. Interestingly, one mutant, while able to adopt the overall postfusion structure, displays significant conformational differences in the vicinity of fusion loops, relative to wild-type gB730. Moreover, this mutant has a diminished ability to associate with liposomes, suggesting that the fusion loops in this mutant have decreased functional activity. We propose that these insertions cause a fusion-deficient phenotype not by preventing conversion of gB to a postfusion-like conformation but rather by interfering with other gB functions.Herpes simplex virus type 1 (HSV-1) is the prototype of the diverse herpesvirus family that includes many notable human pathogens (26). In addition to the icosahedral capsid and the tegument that surround its double-stranded DNA genome, herpesviruses have an envelope—an outer lipid bilayer—bearing a number of surface glycoproteins. During infection, HSV-1 must fuse its envelope with a cellular membrane in order to deliver the capsid into a target host cell. Among its viral glycoproteins, only glycoprotein C (gC), gB, gD, gH, and gL participate in this entry process, and only the last four are required for fusion (28). Although gD is found only in alphaherpesviruses, all herpesviruses encode gB, gH, and gL, which constitute their core fusion machinery. Of these three proteins, gB is the most highly conserved.We recently determined the crystal structure of a nearly full-length ectodomain of HSV-1 gB, gB730 (18). The crystal structure of the ectodomain of gB from Epstein-Barr virus, another herpesvirus, has also been subsequently determined (4). The two structures showed similarities between gB and other viral fusion proteins, in particular, G from an unrelated vesicular stomatitis virus (VSV) (25), leading to the hypothesis that gB is a fusogen, presumably directly involved in bringing the viral and host cell membranes together to enable their fusion. However, gB alone is known to be insufficient for membrane fusion; the gH/gL heterodimer is also required. This insufficiency raises the question of exactly how gB functions during viral entry. Answering this question is critical for understanding the complex mechanism that herpesviruses use to enter their host cells.In acting as a viral fusogen, gB must undergo dramatic conformational changes, refolding through a series of conformational intermediates from its initial, or prefusion form, to its final, or postfusion form (15). These conformational changes are not only necessary to bring the two membranes into proximity; they are also thought to provide the energy for the fusion process. The prefusion form corresponds to the protein present on the viral surface prior to initiation of fusion. The postfusion form represents the protein after fusion of the viral and host cell membranes. The available gB structure likely represents its postfusion form, since it shares more in common with the postfusion rather than the prefusion structure of vesicular stomatitis virus (VSV) G (3, 17). However, the prefusion form has not yet been characterized.Recently, a panel of gB mutants was generated by using random linker-insertion mutagenesis (21). Of these mutants, 16 were particularly interesting because they were nonfunctional in cell-cell fusion assays despite being expressed on the cell surface at levels that indicate proper folding for transport. These observations suggested that each insertion somehow interfered with gB function. Insertions in 12 of these mutants are located within the available structure of the gB ectodomain, which allowed Lin and Spear to analyze their locations (21).The most prominent examples of such nonfunctional mutants are two mutants with insertions after residues I185 or E187, henceforth referred to as “cavity mutants” because both I185 and E187 point into a cavity inside the gB trimer (Fig. 1B and D). Although this cavity might accommodate a single 5-amino-acid insertion, it “is not large enough to accommodate three 5-amino-acid insertions” (21) that would be present in the trimer (one insertion per protomer).Open in a separate windowFIG. 1.Location of the insertion sites in the sequence of gB and the structure of the postfusion form of its ectodomain. (A) Linear diagram of the full-length gB with functional domains highlighted (as in reference 18). Domain I is shown in cyan, domain II in green, domain III in yellow, domain IV in orange, domain V in red, and the disordered region between domains II and III in purple. Regions absent from the crystal structure of gB730 are shown in gray. Sequences in the region of 5-amino-acid insertions (residues 181 to 190 and residues 661 to 680) are shown in black. Arrows mark the locations of 5-amino-acid insertions, shown as red text. (B) Crystal structure of gB730 (18). Residues preceding the 5-amino-acid insertions in mutants studied here are shown as spheres colored by domain, consistent with panel A. Boxes delineate the hinge region, enlarged in panel C, and the cavity region, enlarged in panel D. (C) Close-up view of the hinge region shown in molecular surface representation, with residues 663 to 675 displayed as sticks. Hydrophobic residues are colored orange. Residues preceding the 5-amino-acid insertions in mutants studied here are labeled with asterisks; remaining labels correspond to additional hydrophobic residues in the 663-675 region. (D) Enlarged view of the cavity region. Residues that line the cavity and are not solvent exposed are colored magenta. Residue E187 of each protomer is colored teal and shown as spheres. Fusion loops for two protomers are marked with asterisks; the third pair of fusion loops lies behind the crystal structure and is not visible. Panels B, C, and D were made by using Pymol (http://www.pymol.org/).Five other nonfunctional mutants have insertions after residues D663, T665, V667, I671, or L673, respectively. We refer to them as “hinge mutants.” These residues lie in the region located between domains IV and V, which has been termed the hinge region because it may play an important role during the conformational transition from the prefusion to the postfusion form (17). Lin and Spear proposed that insertions following these residues “would likely affect hinge regions” (21), with the implication that they may prevent gB from refolding into the postfusion conformation. Our analysis suggested that insertions after these residues could, perhaps, be sterically accommodated in the structure but would probably be energetically unfavorable by causing several buried hydrophobic side chains in the 665-673 region, such as F670, I671, and L673, to become exposed (Fig. 1B and C).In light of these observations, we hypothesized that the insertion mutants are “trapped” in a prefusion form. We decided to test this hypothesis by determining whether the ectodomain of gB containing one of these insertion mutations is able to assume the conformation seen in the crystal structure of the wild-type gB ectodomain, which we are referring to as the likely postfusion conformation. For this purpose, we chose one cavity mutant, containing an insertion after E187, and four hinge mutants, containing insertions after T665, V667, I671, or L673, respectively. We chose to test four hinge mutants because structure analysis suggested to us that insertions following the respective residues might not affect the structure in precisely the same way. We expressed the soluble ectodomain of each mutant by using a baculovirus expression system and characterized the purified proteins by using biochemical and biophysical methods. Surprisingly, we found that the ectodomains of all five mutants assume a conformation similar to that of the wild-type gB ectodomain. The four hinge mutants had biochemical properties and overall three-dimensional structures that were indistinguishable from those of the wild-type gB ectodomain. We conclude that these mutants undergo only minor local conformational changes to relieve the steric strain resulting from the presence of 5 extra amino acids. Interestingly, the cavity mutant, while able to adopt the overall postfusion structure, still displayed significant conformational differences relative to wild-type gB. Because these conformational differences are in the vicinity of fusion loops, we conclude that the fusion loops in this mutant have decreased functional activity.