Glycoprotein B (gB) enables the fusion of viral and cell membranes during access of herpesviruses. was generated by using random 5-amino-acid-linker insertion mutagenesis. Several mutants were unable to mediate cell-cell fusion despite becoming expressed within the cell surface. Mapping of the insertion sites onto the crystal structure of gB730 suggested that several insertions is probably not accommodated in the postfusion form. Therefore we hypothesized that some insertion mutants were nonfunctional due to being “caught” inside a prefusion form. Here we generated five insertion mutants as soluble ectodomains and characterized them biochemically. We display the ectodomains of all five mutants presume conformations similar to that of the wild-type gB730. Four mutants have biochemical properties and overall constructions 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 variations in the vicinity of fusion loops relative to wild-type gB730. Moreover this mutant has a diminished ability to associate with liposomes suggesting the fusion loops with this mutant have decreased practical 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 additional gB functions. Herpes simplex virus type 1 (HSV-1) is the prototype of the varied herpesvirus family that includes many notable human being 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 CAV1 bilayer-bearing a number of surface glycoproteins. During illness HSV-1 must fuse its envelope having a cellular membrane in order to deliver the SCR7 capsid into a target sponsor cell. Among its viral SCR7 glycoproteins only glycoprotein C (gC) gB gD gH and gL participate in this access 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 identified 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 computer virus another herpesvirus has also been subsequently decided (4). The two structures showed similarities between gB and additional viral SCR7 fusion proteins in particular SCR7 G from an unrelated vesicular stomatitis computer virus (VSV) (25) leading to the hypothesis that gB is definitely a fusogen presumably directly involved in bringing the viral and sponsor cell membranes collectively to enable their fusion. However gB only is known to become insufficient for membrane fusion; the gH/gL heterodimer is also required. This insufficiency increases the query of exactly how gB functions during viral access. Answering this query is critical for understanding the complex mechanism that SCR7 herpesviruses use to enter their sponsor cells. In acting like 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 within the viral surface prior to initiation of fusion. The postfusion form represents the protein after fusion of the viral and sponsor 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 SCR7 of vesicular stomatitis computer 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 becoming expressed within the cell surface at levels that indicate appropriate folding for transport. These observations suggested that every insertion somehow interfered with gB function. Insertions in 12 of these mutants are located within the available structure of.