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 Low pH-Induced Conformational Change in Herpes Simplex Virus Glycoprotein B

2010 ◽  
Vol 84 (8) ◽  
pp. 3759-3766 ◽  
Author(s):  
Stephen J. Dollery ◽  
Mark G. Delboy ◽  
Anthony V. Nicola
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Conformational Changes ◽  
Low Ph ◽  
Host Cells ◽  
Glycoprotein B ◽  
Antigenic Structure ◽  
Acidic Ph ◽  
Herpes Simplex Virus Glycoprotein ◽  
Simplex Virus

ABSTRACT Herpesviruses can enter host cells using pH-dependent endocytosis pathways in a cell-specific manner. Envelope glycoprotein B (gB) is conserved among all herpesviruses and is a critical component of the complex that mediates membrane fusion and entry. Here we demonstrate that mildly acidic pH triggers specific conformational changes in herpes simplex virus (HSV) gB. The antigenic structure of gB was specifically altered by exposure to low pH both in vitro and during entry into host cells. The oligomeric conformation of gB was altered at a similar pH range. Exposure to acid pH appeared to convert virion gB into a lower-order oligomer. The detected conformational changes were reversible, similar to those in other class III fusion proteins. Exposure of purified, recombinant gB to mildly acidic pH resulted in similar changes in conformation and caused gB to become more hydrophobic, suggesting that low pH directly affects gB. We propose that intracellular low pH induces alterations in gB conformation that, together with additional triggers such as receptor binding, are essential for virion-cell fusion during herpesviral entry by endocytosis.

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 Mutations in the Herpes Simplex Virus 1 Glycoprotein B Ectodomain Arm Impart a Slow-Entry Phenotype

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Qing Fan ◽  
Sarah J. Kopp ◽  
Sarah A. Connolly ◽  
Richard Longnecker
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Bacterial Artificial Chromosome ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Elevated Temperatures ◽  
Artificial Chromosome ◽  
Glycoprotein B ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus

ABSTRACTGlycoprotein B (gB) is the conserved herpesvirus fusion protein, and it is required for the entry of herpesviruses. The structure of the postfusion conformation of gB has been solved for several herpesviruses; however, the gB prefusion crystal structure and the details of how the protein refolds from a prefusion to a postfusion form to mediate fusion have not been determined. Using structure-based mutagenesis, we previously reported that three mutations (I671A, H681A, and F683A) in the C-terminal arm of the gB ectodomain greatly reduced cell-cell fusion. This fusion deficit could be rescued by the addition of a hyperfusogenic mutation, suggesting that the gB triple mutant was not misfolded. Using a bacterial artificial chromosome (BAC), we constructed two independent herpes simplex virus 1 mutant strains (gB 3A) carrying the three arm mutations. The gB 3A viruses have 200-fold smaller plaques than the wild-type virus and demonstrate remarkably delayed entry into cells. Single-step and multistep growth curves show that gB 3A viruses have delayed replication kinetics. Interestingly, incubation at 40°C promoted the entry of the gB 3A viruses. We propose that the gB 3A viruses’ entry deficit is due to a loss of interactions between residues in the gB C-terminal arm and the coiled-coil core of gB. The results suggest that the triple alanine mutation may destabilize the postfusion gB conformation and/or stabilize the prefusion gB conformation and that exposure to elevated temperatures can overcome the defect in gB 3A viruses.IMPORTANCEBecause of its complexity, the mechanism of herpesvirus entry into cells is not well understood. Our study investigated one of the most important unanswered questions about herpesvirus entry; i.e., how does the herpesvirus fusion protein gB mediate membrane fusion? gB is an essential protein that is conserved in all herpesviruses and is thought to undergo a conformational change to provide the energy to fuse the viral and cellular membranes. Using our understanding of the structure of gB, we designed mutations in the gB “arm” region that we predicted would impede gB function. We introduced these mutations into herpes simplex virus 1 by using a bacterial artificial chromosome, and the mutant virus exhibited a drastically delayed rate of entry. This entry defect was rescued by incubation at elevated temperatures, supporting a hypothesis that the engineered mutations altered the energetics of gB refolding. This study supports our hypothesis that an interaction between the gB arm and the core of gB is critical for gB refolding and the execution of membrane fusion, thus providing key details about the function of gB in herpesvirus-mediated fusion and subsequent virus entry.

Evidence for a Role of the Membrane-Proximal Region of Herpes Simplex Virus Type 1 Glycoprotein H in Membrane Fusion and Virus Inhibition

ChemBioChem ◽  
2007 ◽  
Vol 8 (8) ◽  
pp. 885-895 ◽  
Author(s):  
Stefania Galdiero ◽  
Annarita Falanga ◽  
Mariateresa Vitiello ◽  
Marina D'Isanto ◽  
Craig Collins ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Membrane Fusion ◽  
Proximal Region ◽  
Virus Inhibition ◽  
Glycoprotein H ◽  
Simplex Virus ◽  
Membrane Proximal Region

scholarly journals Herpes Simplex Virus 1 Glycoprotein B and US3 Collaborate To Inhibit CD1d Antigen Presentation and NKT Cell Function

2011 ◽  
Vol 85 (16) ◽  
pp. 8093-8104 ◽  
Author(s):  
P. Rao ◽  
H. T. Pham ◽  
A. Kulkarni ◽  
Y. Yang ◽  
X. Liu ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Antigen Presentation ◽  
Cell Function ◽  
Glycoprotein B ◽  
Nkt Cell ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus

scholarly journals Functional and Physical Interactions of the Herpes Simplex Virus Type 1 UL20 Membrane Protein with Glycoprotein K

2008 ◽  
Vol 82 (13) ◽  
pp. 6310-6323 ◽  
Author(s):  
Timothy P. Foster ◽  
Vladimir N. Chouljenko ◽  
K. G. Kousoulas
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Herpes Simplex Virus Type ◽  
Protein Protein Interactions ◽  
Glycoprotein K ◽  
Er Retention ◽  
Simplex Virus

ABSTRACT Herpes simplex virus type 1 glycoprotein K (gK) and the UL20 protein (UL20p) are coordinately transported to the trans-Golgi network (TGN) and cell surfaces and are required for cytoplasmic virion envelopment at the TGN. In addition, cell surface expression of gK and UL20p is required for virus-induced cell fusion. Previously, confocal microscopy colocalization and intracellular transport experiments strongly suggested direct protein-protein interactions between gK and UL20p. Direct protein-protein interactions between gK and UL20p were demonstrated through reciprocal coimmunoprecipitation experiments, as well as with glutathione S-transferase (GST) pull-down experiments. A fusion protein consisting of the amino-terminal 66 amino acids of UL20p fused in-frame with GST was expressed in Escherichia coli and purified via glutathione column chromatography. Precipitation of GST-UL20p from mixtures of GST-UL20p fusion protein with cellular extracts containing gK specifically coprecipitated gK but not other viral glycoproteins. The purified UL20p-GST fusion protein reacted with all gK-associated protein species. It was concluded that the amino terminus of UL20p, most likely, interacted with gK domain III, which is predicted to lie intracellularly. UL20p-gK domain-specific interactions must serve important functions in the coordinate transport of UL20p and gK to the TGN, because retention of UL20p in the endoplasmic reticulum (ER) via the addition of an ER retention signal at the carboxyl terminus of UL20p forced the ER retention of gK and drastically inhibited intracellular virion envelopment and virus-induced cell fusion.

scholarly journals The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Juan Fontana ◽  
Doina Atanasiu ◽  
Wan Ting Saw ◽  
John R. Gallagher ◽  
Reagan G. Cox ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Protein ◽  
Surface Protein ◽  
Skin Lesions ◽  
Enveloped Viruses ◽  
Host Membrane ◽  
Simplex Virus ◽  
Viral Surface Protein ◽  
Viral Surface

ABSTRACTAll enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an initial prefusion conformation to a final postfusion conformation. Strikingly, the prefusion conformation of the herpesvirus fusion protein, gB, is poorly understood. Herpes simplex virus (HSV), a model system for herpesviruses, causes diseases ranging from mild skin lesions to serious encephalitis and neonatal infections. Using cryo-electron tomography and subtomogram averaging, we have characterized the structure of the prefusion conformation and fusion intermediates of HSV-1 gB. To this end, we have set up a system that generates microvesicles displaying full-length gB on their envelope. We confirmed proper folding of gB by nondenaturing electrophoresis-Western blotting with a panel of monoclonal antibodies (MAbs) covering all gB domains. To elucidate the arrangement of gB domains, we labeled them by using (i) mutagenesis to insert fluorescent proteins at specific positions, (ii) coexpression of gB with Fabs for a neutralizing MAb with known binding sites, and (iii) incubation of gB with an antibody directed against the fusion loops. Our results show that gB starts in a compact prefusion conformation with the fusion loops pointing toward the viral membrane and suggest, for the first time, a model for gB’s conformational rearrangements during fusion. These experiments further illustrate how neutralizing antibodies can interfere with the essential gB structural transitions that mediate viral entry and therefore infectivity.IMPORTANCEThe herpesvirus family includes herpes simplex virus (HSV) and other human viruses that cause lifelong infections and a variety of diseases, like skin lesions, encephalitis, and cancers. As enveloped viruses, herpesviruses must fuse their envelope with the host membrane to start an infection. This process is mediated by a viral surface protein that transitions from an initial conformation (prefusion) to a final, more stable, conformation (postfusion). However, the prefusion conformation of the herpesvirus fusion protein (gB) is poorly understood. To elucidate the structure of the prefusion conformation of HSV type 1 gB, we have employed cryo-electron microscopy to study gB molecules expressed on the surface of vesicles. Using different approaches to label gB’s domains allowed us to model the structures of the prefusion and intermediate conformations of gB. Overall, our findings enhance our understanding of HSV fusion and lay the groundwork for the development of new ways to prevent and block HSV infection.

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