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

Glycoprotein L (gL) is one of four glycoproteins required for the entry of herpes simplex virus (HSV) into cells and for virus-induced cell fusion. This glycoprotein oligomerizes with gH to form a membrane-bound heterodimer but can be secreted when expressed without gH. Twelve unique gL linker-insertion mutants were generated to identify regions critical for gH binding and gH/gL processing and regions essential for cell fusion and viral entry. All gL mutants were detected on the cell surface in the absence of gH, suggesting incomplete cleavage of the signal peptide or the presence of a cell surface receptor for secreted gL. Coexpression with gH enhanced the levels of cell surface gL detected by antibodies for all gL mutants except those that were defective in their interactions with gH. Two insertions into a conserved region of gL abrogated the binding of gL to gH and prevented gH expression on the cell surface. Three other insertions reduced the cell surface expression of gH and/or altered the properties of gH/gL heterodimers. Altered or absent interaction of gL with gH was correlated with reduced or absent cell fusion activity and impaired complementation of virion infectivity. These results identify a conserved domain of gL that is critical for its binding to gH and two noncontiguous regions of gL, one of which contains the conserved domain, that are critical for the gH/gL complex to perform its role in membrane fusion.Glycoprotein L (gL) is one of the four glycoproteins required for the entry of herpes simplex virus (HSV) into cells and for virus-induced cell fusion (26, 33). The others are gB, gD, and gH (30). The functional unit containing gL is a heterodimer formed with gH (gH/gL) (15). Because mature gL has no membrane-spanning domain, other than a cleavable signal peptide, it is secreted unless it is coexpressed with gH, a type 1 glycoprotein that anchors gL to the cell membrane (2). Also, gH is not properly processed or transported out of the endoplasmic reticulum unless it is coexpressed with gL (15).Most, if not all, herpesviruses express orthologs of gB, gH, and gL, which are believed to form the core membrane-fusing machinery necessary for viral entry and cell fusion. For some herpesviruses, such as Epstein-Barr virus and human cytomegalovirus, the gH/gL oligomer may contain additional viral subunits that can influence binding of the complex to cell receptors and determine cell tropism (14, 34, 35). For HSV, however, only gD and gB have been shown to have receptor-binding activities that are required for entry (27, 31). Although HSV gH has an RGD motif and the gH/gL heterodimer can bind to certain integrins, this binding seems not to be necessary for viral entry (3, 22).The initial interaction of HSV with cells can be the reversible attachment of virus to cell surface heparan sulfate, mediated by viral glycoprotein gB and/or gC (29). Then, gD can bind to one of its receptors, including herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor receptor family; nectin-1 or nectin-2, cell adhesion molecules belonging to the immunoglobulin superfamily; or specific sites in heparan sulfate generated by 3-O-sulfotransferases (31). In addition to binding to heparan sulfate, gB can also bind to other cell surface receptors, including paired immunoglobulin-like receptor alpha (PILRα) (27). Binding of both gD and gB to one of their respective receptors appears to be required for triggering the membrane-fusing activity of gB and/or gH/gL, which leads to viral entry.A recent X-ray structure of HSV type 1 (HSV-1) gB suggests that it is a class III viral fusogen similar in domain organization, but not primary sequence, to the G protein of vesicular stomatitis virus (13). It has been proposed that HSV-1 gH has features characteristic of class I viral fusogens, such as putative heptad repeats and fusion peptides (6, 9-11). Also, peptides matching the sequence of gH can interact with lipids and/or induce the fusion of lipid vesicles (4, 5, 8). Hemifusion between cells and between virus and cell can be induced by gH/gL and gD in the absence of gB (32). Many questions remain about the respective roles of gH/gL and gB in inducing membrane fusion.The four conserved cysteines in gL were found to be essential for gL-gH association and function (1). Mutational analyses of gL by C-terminal deletions showed that the first 147 amino acids of gL are sufficient for association with gH but that the first 161 amino acids are necessary for cotransport of gH and gL to the cell surface (17, 23) and for gL activity in cell fusion and viral entry (17). Lastly, certain anti-gL monoclonal antibodies (MAbs) can inhibit cell fusion but not viral entry, despite demonstrable binding of the MAbs to virus, suggesting that gL may play a different role in each process (21). These MAbs were mapped to the C-terminal region of gL (21, 23). The diagram at the bottom of Fig. ​Fig.11 shows the locations of the gL features mentioned above and of the signal peptide.Open in a separate windowFIG. 1.Effects of insertional mutations on HSV-1 gL and gH cell surface expression. CHO cells were transfected with plasmids expressing gH and WT gL or a gL mutant. Cell surface expression of gL and gH was quantified by CELISA using polyclonal R88 antiserum (filled circles) and MAb 52S (open triangles), respectively. A linear representation of the gL polypeptide is shown below the graph, with coded bars identifying features of gL. The bars represent the signal peptide (uncolored hatched), the N-terminal 161-amino-acid fragment necessary for the formation of functional gH/gL complexes (dark gray), highly conserved residues within this fragment (cross-hatched dark- gray bar), and epitopes recognized by a panel of anti-gL MAbs (light-gray and uncolored vertically striped bars). The values presented for cell surface expression of each mutant gL and of cotransfected WT gH are means from three independent experiments expressed as percentages of WT gL (or of gH cotransfected with WT gL) values, after subtraction of background values obtained in the absence of gL expression and as a function of the position of the insertion. Standard deviations are presented in Fig. ​Fig.22 and ​and33 for similar experiments.The interactions between gL and gH required for proper intracellular transport, processing, and cell surface expression make it difficult to investigate the functional role of one of these glycoproteins in cell fusion and viral entry independently of the other. We generated a panel of gL linker-insertion mutants to identify regions critical for gH binding and transport and regions essential for cell fusion and viral entry. One aim was to determine whether these roles of gL could be dissociated or were linked. Characterization of 12 unique gL linker-insertion mutants showed that (i) a conserved domain of gL is critical for the physical interaction of gL with gH and for the normal processing of gH, (ii) two noncontiguous regions of gL, one of which contains the highly conserved domain, are critical for the normal conformation and function of gH/gL heterodimers, and (iii) wild-type (WT) and mutant gLs can be detected on the cell surface in the absence of gH, suggesting the possibility of an independent role for uncomplexed gL. These results support and extend previous studies suggesting that gL has a larger role in membrane fusion than serving as a chaperone for gH and that specific mutations in gL can influence the function of the gH/gL heterodimer.