Mutations in the Amino Terminus of Herpes Simplex Virus Type 1 gL Can Reduce Cell-Cell Fusion without Affecting gH/gL Trafficking

Abstract

The gH/gL heterodimer represents two of the four herpes simplex virus glycoproteins necessary and sufficient for membrane fusion. We generated deletions and point mutations covering gL residues 24 to 43 to investigate that region's role in gH/gL intracellular trafficking and in membrane fusion. Multiple mutants displayed a 40 to 60% reduction in cell fusion with no effect on gH/gL trafficking. The amino terminus of gL plays an important role in the gH/gL contribution to membrane fusion. Herpes simplex virus (HSV) is enveloped by a lipid bilayer containing approximately a dozen or more envelope glycoproteins. The virus relies upon its envelope glycoproteins to fuse the viral membrane with a cellular membrane to gain access to a host cell. The envelope glycoproteins that are necessary and sufficient to mediate membrane fusion are glycoproteins B, D, H, and L (gB, gD, gH, and gL) (1-3). The smallest of the HSV fusion glycoproteins is gL, which comprises 224 amino acids, with no obvious transmembrane domain. The integral membrane protein gH binds gL, probably in the endoplasmic reticulum (ER), and the corresponding gH/gL heterodimer then transits to sites of viral envelopment and the plasma membrane (4, 5). An intact HSV type 1 (HSV-1) gH will not exit the ER unless it is bound to gL (4-7). The gH/gL heterodimer has been postulated to have the hallmarks of a viral fusion protein and to play a direct role in membrane fusion (8-13). However, the HSV-2 gH/gL structure does not resemble any known viral fusion protein, and there is recent evidence that HSV gH/gL plays more of a regulatory and/or structural role in membrane fusion executed by the class III fusion protein gB (14-17).The crystal structure has been determined for the HSV-2 gH (residues 48 to 803)/gL (residues 20 to 224) heterodimer (16). Three domains, H1 to H3, were assigned to the gH structure, with domain H1 further subdivided into H1A and H1B (16). The H1A and H1B subdomains (residues 49 to 115 and 13 to 327, respectively) form a vise to clamp onto gL. The majority of gL does not adopt an identifiable secondary structure, with three helices and two ␤-sheets comprising only 30% of gL residues. There are extensive areas of contact between gL and gH, such that most gL residues interact with or are in very close proximity to subdomains H1A and H1B. Regions of gL that extend outward from the gH/gL heterodimer and do not appear to interact with gH include residues at the amino terminus (residues 26 to 44) and a small ␤-strand near the carboxy terminus, residues 197 to 203 (16).To investigate gH/gL trafficking and function in membrane fusion by using targeted mutagenesis, we were most interested in regions predicted by the crystal structure that project outwards. We adopted this strategy to minimize the chance we would generate gH or gL mutants that did not stably associate, because such mutants would be unlikely to provide information on gH/gL function in membrane fusion. HSV-1 gL residues 162 to 224 (including ␤-strand residues 197 to 203) have been deleted i...

Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes

“…A role for the N-terminal region of gH/gL in receptor binding is consistent with what has been observed in other herpesvirus gH/gLs (39)(40)(41)(42)(43). In the alpha herpesvirus PrV, a fusion protein in which the ectodomain of the receptor-binding protein gD was fused to the N-terminal region of gH was able to interact with gB for entry, substituting for gD, gH, and gL (44).…”

Using Split Luciferase Assay and Anti-Herpes Simplex Virus Glycoprotein Monoclonal Antibodies To Predict a Functional Binding Site between gD and gH/gL

“…CHL27 recognizes a linear epitope (gH 37-47) within the flexible N terminus of gH (Table 1) (6). In contrast, 53S targets a conformational epitope that requires both gH and gL (18)(19)(20)(21)(22) and includes gL residues P48 (21) and P77 (6). Figure 5H shows that the combination of CHL27 and 53S was very efficient and that the effect was additive, consistent with the mapping data.…”

“…The second region of contact is defined by MC14 (gD 262-272) and CHL18 (gL 209-218). Because Mab CHL17 (gH [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] showed an additive inhibitory effect with both MC5 and MC14, the CHL17 epitope belongs to a third contact site on gH/gL. Interestingly, CHL17 and CHL27 have distinct mechanisms of action despite the adjacent location of their epitopes.…”

“…Remarkably, the three sites in gH/gL defined by CHL17, CHL27, and CHL18 epitopes are in flexible regions (17,22,(37)(38)(39)(40)(41) at the gH N terminus and the gL C terminus and might have a role in the tethering of gD in the complex. Whether these flexible regions move in response to the receptor-activated gD or as a consequence of gD binding to gH/gL is yet unknown.…”

Scanning Mutagenesis of Human Cytomegalovirus Glycoprotein gH/gL

“…In contrast to that view, other analyses of HSV, pseudorabies virus (PRV), and EBV have suggested a more direct role for gL in the fusion function of gH/gL (37)(38)(39)(40). The solved crystal structures of EBV and HSV gH/gL provided important insights by showing a very intimate association between gH and gL (25,26).…”