scholarly journals Herpes Simplex Virus Glycoprotein B Associates with Target Membranes via Its Fusion Loops

2009 ◽  
Vol 83 (13) ◽  
pp. 6825-6836 ◽  
Author(s):  
Brian P. Hannah ◽  
Tina M. Cairns ◽  
Florent C. Bender ◽  
J. Charles Whitbeck ◽  
Huan Lou ◽  
...  
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Lipid Membranes ◽  
Virus Entry ◽  
Expression System ◽  
Lipid Binding ◽  
Mutant Proteins ◽  
Herpes Simplex Virus Glycoprotein ◽  
Simplex Virus

ABSTRACT Herpes simplex virus (HSV) glycoproteins gB, gD, and gH/gL are necessary and sufficient for virus entry into cells. Structural features of gB are similar to those of vesicular stomatitis virus G and baculovirus gp64, and together they define the new class III group of fusion proteins. Previously, we used mutagenesis to show that three hydrophobic residues (W174, Y179, and A261) within the putative gB fusion loops are integral to gB function. Here we expanded our analysis, using site-directed mutagenesis of each residue in both gB fusion loops. Mutation of most of the nonpolar or hydrophobic amino acids (W174, F175, G176, Y179, and A261) had severe effects on gB function in cell-cell fusion and null virus complementation assays. Of the six charged amino acids, mutation of H263 or R264 also negatively affected gB function. To further analyze the mutants, we cloned the ectodomains of the W174R, Y179S, H263A, and R264A mutants into a baculovirus expression system and compared them with the wild-type (WT) form, gB730t. As shown previously, gB730t blocks virus entry into cells, suggesting that gB730t competes with virion gB for a cell receptor. All four mutant proteins retained this function, implying that fusion loop activity is separate from gB-receptor binding. However, unlike WT gB730t, the mutant proteins displayed reduced binding to cells and were either impaired or unable to bind naked, cholesterol-enriched liposomes, suggesting that it may be gB-lipid binding that is disrupted by the mutations. Furthermore, monoclonal antibodies with epitopes proximal to the fusion loops abrogated gB-liposome binding. Taken together, our data suggest that gB associates with lipid membranes via a fusion domain of key hydrophobic and hydrophilic residues and that this domain associates with lipid membranes during fusion.

scholarly journals Structure-Based Analysis of the Herpes Simplex Virus Glycoprotein D Binding Site Present on Herpesvirus Entry Mediator HveA (HVEM)

2002 ◽  
Vol 76 (21) ◽  
pp. 10894-10904 ◽  
Author(s):  
Sarah A. Connolly ◽  
Daniel J. Landsburg ◽  
Andrea Carfi ◽  
Don C. Wiley ◽  
Roselyn J. Eisenberg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Binding Site ◽  
Virus Entry ◽  
Cell Surface Receptor ◽  
Glycoprotein D ◽  
Herpes Simplex Virus Glycoprotein ◽  
Contact Residues ◽  
Simplex Virus

ABSTRACT Binding of herpes simplex virus (HSV) envelope glycoprotein D (gD) to a cell surface receptor is an essential step of virus entry. We recently determined the crystal structure of gD bound to one receptor, HveA. HveA is a member of the tumor necrosis factor receptor family and contains four characteristic cysteine-rich domains (CRDs). The first two CRDs of HveA are necessary and sufficient for gD binding. The structure of the gD-HveA complex reveals that 17 amino acids in HveA CRD1 and 4 amino acids in HveA CRD2 directly contact gD. To determine the contribution of these 21 HveA residues to virus entry, we constructed forms of HveA mutated in each of these contact residues. We determined the ability of the mutant proteins to bind gD, facilitate virus entry, and form HveA oligomers. Our results point to a binding hot spot centered around HveA-Y23, a residue that protrudes into a crevice on the surface of gD. Both the hydroxyl group and phenyl group of HveA-Y23 contribute to HSV entry. Our results also suggest that an intermolecular β-sheet formed between gD and HveA residues 35 to 37 contributes to binding and that a C37-C19 disulfide bond in CRD1 is a critical component of HveA structure necessary for gD binding. The results argue that CRD2 is required for gD binding mainly to provide structural support for a gD binding site in CRD1. Only one mutant, HveA-R75A, exhibited enhanced gD binding. While some mutations influenced complex formation, the majority did not affect HSV entry, suggesting that most contact residues contribute to HveA receptor function collectively rather than individually. This structure-based dissection of the gD-HveA binding site highlights the contribution of key residues within HveA to gD binding and HSV entry and defines a target region for the design of small-molecule inhibitors.

scholarly journals Structure-Based Mutagenesis of Herpes Simplex Virus Glycoprotein D Defines Three Critical Regions at the gD-HveA/HVEM Binding Interface

2003 ◽  
Vol 77 (14) ◽  
pp. 8127-8140 ◽  
Author(s):  
Sarah A. Connolly ◽  
Daniel J. Landsburg ◽  
Andrea Carfi ◽  
Don C. Wiley ◽  
Gary H. Cohen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Entry ◽  
The Other ◽  
Glycoprotein D ◽  
Wild Type ◽  
Binding Interface ◽  
Simplex Virus ◽  
Critical Regions

ABSTRACT Herpes simplex virus (HSV) entry into cells requires the binding of glycoprotein D (gD) to one of several cell surface receptors. The crystal structure of gD bound to one of these receptors, HveA/HVEM, reveals that the core of gD comprises an immunoglobulin fold flanked by a long C-terminal extension and an N-terminal hairpin loop. HveA is a member of the tumor necrosis factor receptor family and contains four cysteine-rich domains (CRDs) characteristic of this family. Fourteen amino acids within the gD N-terminal loop comprise the entire binding site for HveA. To determine the contribution of each gD contact residue to virus entry, we constructed gD molecules mutated in these amino acids. We determined the abilities of the gD mutants to bind receptors, facilitate virus entry, and mediate cell-cell fusion. Seven of the gD mutants exhibited wild-type levels of receptor binding and gD function. Results from the other seven gD mutants revealed three critical regions at the gD-HveA interface. (i) Several gD residues that participate in an intermolecular β-sheet with HveA were found to be crucial for HveA binding and entry into HveA-expressing cells. (ii) Two gD residues that contact HveA-Y23 contributed to HveA binding but were not required for mediating entry into cells. HveA-Y23 fits into a crevice on the surface of gD and was previously shown to be essential for gD binding. (iii) CRD2 was previously shown to contribute to gD binding, and this study shows that one gD residue that contacts CRD2 contributes to HveA binding. None of the gD mutations prevented interaction with nectin-1, another gD receptor. However, when cotransfected with the other glycoproteins required for fusion, two gD mutants gained the ability to mediate fusion of cells expressing nectin-2, a gD receptor that interacts with several laboratory-derived gD mutants but not with wild-type gD. Thus, results from this panel of gD mutants as well as those of previous studies (A. Carfi, S. H. Willis, J. C. Whitbeck, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and D. C. Wiley, Mol. Cell 8:169-179, 2001, and S. A. Connolly, D. J. Landsburg, A. Carfi, D. C. Wiley, R. J. Eisenberg, and G. H. Cohen, J. Virol. 76:10894-10904, 2002) provide a detailed picture of the gD-HveA interface and the contacts required for functional interaction. The results demonstrate that of the 35 gD and HveA contact residues that comprise the gD-HveA interface, only a handful are critical for complex formation.

scholarly journals Mutational Evidence of Internal Fusion Loops in Herpes Simplex Virus Glycoprotein B

2007 ◽  
Vol 81 (9) ◽  
pp. 4858-4865 ◽  
Author(s):  
Brian P. Hannah ◽  
Ekaterina E. Heldwein ◽  
Florent C. Bender ◽  
Gary H. Cohen ◽  
Roselyn J. Eisenberg
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Proteins ◽  
Glycoprotein B ◽  
Functional Domain ◽  
Virus Glycoprotein ◽  
Herpes Simplex Virus Glycoprotein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is one of four glycoproteins necessary and sufficient for HSV cellular entry. Recently, the crystal structures of HSV-1 gB and vesicular stomatitis virus glycoprotein G were determined. Surprisingly, the two proteins share remarkable structural homology. Both proteins are homotrimeric and center about a long alpha-helix, features reminiscent of class I fusion proteins, such as influenza virus hemagglutinin or paramyxovirus F. However, these structures revealed that G has internal fusion loops, similar to the fusion loops of the class II fusion proteins, and that these loops are structurally conserved in gB. To examine whether these putative fusion loops are important for gB function, we mutated potential membrane-interacting (hydrophobic) residues to charged amino acids. Of most interest were mutant gB proteins that were expressed on the cell surface and were recognized by monoclonal antibodies against conformational epitopes but lacked the ability to function in cell-cell fusion assays. We find that three of the five hydrophobic amino acids targeted in these loops, tryptophan 174, tyrosine 179, and alanine 261, are integral in the function of gB. Our data suggest that they are part of an important functional domain. We hypothesize that two loops in domain 1 of HSV gB function as fusion loops. Our data are further evidence that gB is a viral fusogen and suggest clues as to how gB may function.

scholarly journals The Membrane-Proximal Region (MPR) of Herpes Simplex Virus gB Regulates Association of the Fusion Loops with Lipid Membranes

mBio ◽  
2012 ◽  
Vol 3 (6) ◽  
Author(s):  
Spencer S. Shelly ◽  
Tina M. Cairns ◽  
J. Charles Whitbeck ◽  
Huan Lou ◽  
Claude Krummenacher ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Protein ◽  
Lipid Membranes ◽  
Expression System ◽  
Proximal Region ◽  
Host Cells ◽  
Glycoprotein B ◽  
Simplex Virus ◽  
Membrane Proximal Region

ABSTRACTGlycoprotein B (gB), gD, and gH/gL constitute the fusion machinery of herpes simplex virus (HSV). Prior studies indicated that fusion occurs in a stepwise fashion whereby the gD/receptor complex activates the entire process, while gH/gL regulates the fusion reaction carried out by gB. Trimeric gB is a class III fusion protein. Its ectodomain of 773 amino acids contains a membrane-proximal region (MPR) (residues 731 to 773) and two fusion loops (FLs) per protomer. We hypothesized that the highly hydrophobic MPR interacts with the FLs, thereby masking them on virions until fusion begins. To test this hypothesis, we made a series of deletion, truncation, and point mutants of the gB MPR. Although the full-length deletion mutants were expressed in transfected cells, they were not transported to the cell surface, suggesting that removal of even small stretches of the MPR was highly detrimental to gB folding. To circumvent this limitation, we used a baculovirus expression system to generate four soluble proteins, each lacking the transmembrane region and cytoplasmic tail. All retained the FLs and decreasing portions of the MPR [gB(773t) (gB truncated at amino acid 773), gB(759t), gB(749t), and gB(739t)]. Despite the presence of the FLs, all were compromised in their ability to bind liposomes compared to the control, gB(730t), which lacks the MPR. We conclude that residues 731 to 739 are sufficient to mask the FLs, thereby preventing liposome association. Importantly, mutation of two aromatic residues (F732 and F738) to alanine restored the ability of gB(739t) to bind liposomes. Our data suggest that the MPR is important for modulating the association of gB FLs with target membranes.IMPORTANCETo successfully cause disease, a virus must infect host cells. Viral infection is a highly regulated, multistep process. For herpesviruses, genetic material transfers from the virus to the target cell through fusion of the viral and host cell lipid membranes. Here, we provide evidence that the ability of the herpes simplex virus (HSV) glycoprotein B (gB) fusion protein to interact with the host membrane is regulated by its membrane-proximal region (MPR), which serves to cover or shield its lipid-associating moieties (fusion loops). This in turn prevents the premature binding of gB with host cells and provides a level of regulation to the fusion process. These findings provide important insight into the complex regulatory steps required for successful herpesvirus infection.

scholarly journals Structural and Antigenic Analysis of a Truncated Form of the Herpes Simplex Virus Glycoprotein gH-gL Complex

1998 ◽  
Vol 72 (7) ◽  
pp. 6092-6103 ◽  
Author(s):  
Tao Peng ◽  
Manuel Ponce de Leon ◽  
Michael J. Novotny ◽  
Hongbin Jiang ◽  
John D. Lambris ◽  
...  
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Infectivity ◽  
Terminal Portion ◽  
Antigenic Analysis ◽  
Amino Terminal ◽  
Herpes Simplex Virus Glycoprotein ◽  
Antigenic Properties ◽  
Simplex Virus

ABSTRACT The herpes simplex virus (HSV) gH-gL complex is essential for virus infectivity and is a major antigen for the host immune system. The association of gH with gL is required for correct folding, cell surface trafficking, and membrane presentation of the complex. Previously, a mammalian cell line was constructed which produces a secreted form of gHt-gL complex lacking the transmembrane and cytoplasmic tail regions of gH. gHt-gL retains a conformation similar to that of its full-length counterpart in HSV-infected cells. Here, we examined the structural and antigenic properties of gHt-gL. We first determined its stoichiometry and carbohydrate composition. We found that the complex consists of one molecule each of gH and gL. The N-linked carbohydrate (N-CHO) site on gL and most of the N-CHO sites on gH are utilized, and both proteins also contain O-linked carbohydrate and sialic acid. These results suggest that the complex is processed to the mature form via the Golgi network prior to secretion. To determine the antigenically active sites of gH and gL, we mapped the epitopes of a panel of gH and gL monoclonal antibodies (MAbs), using a series of gH and gL C-terminal truncation variant proteins produced in transiently transfected mammalian cells. Sixteen gH MAbs (including H6 and 37S) reacted with the N-terminal portion of gH between amino acids 19 and 276. One of the gH MAbs, H12, reacted with the middle portion of gH (residues 476 to 678). Nine gL MAbs (including 8H4 and VIII 62) reacted with continuous epitopes within the C-terminal portion of gL, and this region was further mapped within amino acids 168 to 178 with overlapping synthetic peptides. Finally, plasmids expressing the gH and gL truncations were employed in cotransfection assays to define the minimal regions of both gH and gL required for complex formation and secretion. The first 323 amino acids of gH and the first 161 amino acids of gL can form a stable secreted hetero-oligomer with gL and gH792, respectively, while gH323-gL168 is the smallest secreted hetero-oligomer. The first 648 amino acids of gH are required for reactivity with MAbs LP11 and 53S, indicating that a complex of gH648-gL oligomerizes into the correct conformation. The data suggest that both antigenic activity and oligomeric structure require the amino-terminal portions of gH and gL.

scholarly journals Mutations in the conserved carboxy-terminal hydrophobic region of glycoprotein gB affect infectivity of herpes simplex virus

1999 ◽  
Vol 80 (12) ◽  
pp. 3189-3198 ◽  
Author(s):  
Essam Wanas ◽  
Sue Efler ◽  
Kakoli Ghosh ◽  
Hara P. Ghosh
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Infectivity ◽  
Hydrophobic Region ◽  
Mutant Proteins ◽  
Complementation Assay ◽  
Simplex Virus ◽  
Carboxy Terminal ◽  
Virion Particles

Glycoprotein gB is the most highly conserved glycoprotein in the herpesvirus family and plays a critical role in virus entry and fusion. Glycoprotein gB of herpes simplex virus type 1 contains a hydrophobic stretch of 69 aa near the carboxy terminus that is essential for its biological activity. To determine the role(s) of specific amino acids in the carboxy-terminal hydrophobic region, a number of amino acids were mutagenized that are highly conserved in this region within the gB homologues of the family Herpesviridae. Three conserved residues in the membrane anchor domain, namely A786, A790 and A791, as well as amino acids G743, G746, G766, G770 and P774, that are non-variant in Herpesviridae, were mutagenized. The ability of the mutant proteins to rescue the infectivity of the gB-null virus, K082, in trans was measured by a complementation assay. All of the mutant proteins formed dimers and were incorporated in virion particles produced in the complementation assay. Mutants G746N, G766N, F770S and P774L showed negligible complementation of K082, whereas mutant G743R showed a reduced activity. Virion particles containing these four mutant glycoproteins also showed a markedly reduced rate of entry compared to the wild-type. The results suggest that non-variant residues in the carboxy-terminal hydrophobic region of the gB protein may be important in virus infectivity.

Sign in / Sign up

Export Citation Format

Share Document