scholarly journals Glycoprotein L sets the neutralization profile of murid herpesvirus 4

2009 ◽  
Vol 90 (5) ◽  
pp. 1202-1214 ◽  
Author(s):  
Laurent Gillet ◽  
Marta Alenquer ◽  
Daniel L. Glauser ◽  
Susanna Colaco ◽  
Janet S. May ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Membrane Fusion ◽  
Cell Binding ◽  
Conformation Changes ◽  
Late Endosomes ◽  
Herpesvirus 4 ◽  
Opening Up

Antibodies readily neutralize acute, epidemic viruses, but are less effective against more indolent pathogens such as herpesviruses. Murid herpesvirus 4 (MuHV-4) provides an accessible model for tracking the fate of antibody-exposed gammaherpesvirus virions. Glycoprotein L (gL) plays a central role in MuHV-4 entry: it allows gH to bind heparan sulfate and regulates fusion-associated conformation changes in gH and gB. However, gL is non-essential: heparan sulfate binding can also occur via gp70, and the gB–gH complex alone seems to be sufficient for membrane fusion. Here, we investigated how gL affects the susceptibility of MuHV-4 to neutralization. Immune sera neutralized gL− virions more readily than gL+ virions, chiefly because heparan sulfate binding now depended on gp70 and was therefore easier to block. However, there were also post-binding effects. First, the downstream, gL-independent conformation of gH became a neutralization target; gL normally prevents this by holding gH in an antigenically distinct heterodimer until after endocytosis. Second, gL− virions were more vulnerable to gB-directed neutralization. This covered multiple epitopes and thus seemed to reflect a general opening up of the gH–gB entry complex, which gL again normally restricts to late endosomes. gL therefore limits MuHV-4 neutralization by providing redundancy in cell binding and by keeping key elements of the virion fusion machinery hidden until after endocytosis.

scholarly journals In vivo importance of heparan sulfate-binding glycoproteins for murid herpesvirus-4 infection

2009 ◽  
Vol 90 (3) ◽  
pp. 602-613 ◽  
Author(s):  
Laurent Gillet ◽  
Janet S. May ◽  
Philip G. Stevenson
Keyword(s):  
Heparan Sulfate ◽  
Low Dose ◽  
Envelope Glycoproteins ◽  
High Dose ◽  
Cell Binding ◽  
Host Colonization ◽  
Infection Dose ◽  
Herpesvirus 4

Many herpesviruses bind to heparan sulfate (HS). Murid herpesvirus-4 (MuHV-4) does so via its envelope glycoproteins gp70 and gH/gL. MuHV-4 gp150 further regulates an HS-independent interaction to make that HS-dependent too. Cell binding by MuHV-4 virions is consequently strongly HS-dependent. Gp70 and gH/gL show some in vitro redundancy: an antibody-mediated blockade of HS binding by one is well tolerated, whereas a blockade of both severely impairs infection. In order to understand the importance of HS binding for MuHV-4 in vivo, we generated mutants lacking both gL and gp70. As expected, gL−gp70− MuHV-4 showed very poor cell binding. It infected mice at high dose but not at low dose, indicating defective host entry. But once entry occurred, host colonization, which for MuHV-4 is relatively independent of the infection dose, was remarkably normal. The gL−gp70− entry deficit was much greater than that of gL− or gp70− single knockouts. And gp150 disruption, which allows HS-independent cell binding, largely rescued the gL−gp70− cell binding and host entry deficits. Thus, it appeared that MuHV-4 HS binding is important in vivo, principally for efficient host entry.

scholarly journals Glycoprotein B switches conformation during murid herpesvirus 4 entry

2008 ◽  
Vol 89 (6) ◽  
pp. 1352-1363 ◽  
Author(s):  
Laurent Gillet ◽  
Susanna Colaco ◽  
Philip G. Stevenson
Keyword(s):  
Neutralizing Antibodies ◽  
Structural Changes ◽  
Glycoprotein B ◽  
Cell Binding ◽  
Glycoprotein G ◽  
Late Endosomes ◽  
Endosomal Acidification ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Herpesvirus 4

Herpesviruses are ancient pathogens that infect all vertebrates. The most conserved component of their entry machinery is glycoprotein B (gB), yet how gB functions is unclear. A striking feature of the murid herpesvirus 4 (MuHV-4) gB is its resistance to neutralization. Here, we show by direct visualization of infected cells that the MuHV-4 gB changes its conformation between extracellular virions and those in late endosomes, where capsids are released. Specifically, epitopes on its N-terminal cell-binding domain become inaccessible, whilst non-N-terminal epitopes are revealed, consistent with structural changes reported for the vesicular stomatitis virus glycoprotein G. Inhibitors of endosomal acidification blocked the gB conformation switch. They also blocked capsid release and the establishment of infection, implying that the gB switch is a key step in entry. Neutralizing antibodies could only partially inhibit the switch. Their need to engage a less vulnerable, upstream form of gB, because its fusion form is revealed only in endosomes, helps to explain why gB-directed MuHV-4 neutralization is so difficult.

scholarly journals A mechanistic basis for potent, glycoprotein B-directed gammaherpesvirus neutralization

2011 ◽  
Vol 92 (9) ◽  
pp. 2020-2033 ◽  
Author(s):  
Daniel L. Glauser ◽  
Anne-Sophie Kratz ◽  
Laurent Gillet ◽  
Philip G. Stevenson
Keyword(s):  
Membrane Fusion ◽  
Neutralizing Antibodies ◽  
Antibody Binding ◽  
Glycoprotein B ◽  
Conformation Change ◽  
Essential Component ◽  
Late Endosomes ◽  
Mechanistic Basis ◽  
Herpesvirus 4

Glycoprotein B (gB) is a conserved, essential component of gammaherpes virions and so potentially vulnerable to neutralization. However, few good gB-specific neutralizing antibodies have been identified. Here, we show that murid herpesvirus 4 is strongly neutralized by mAbs that recognize an epitope close to one of the gB fusion loops. Antibody binding did not stop gB interacting with its cellular ligands or initiating its fusion-associated conformation change, but did stop gB resolving stably to its post-fusion form, and so blocked membrane fusion to leave virions stranded in late endosomes. The conservation of gB makes this mechanism a possible general route to gammaherpesvirus neutralization.

scholarly journals Virion endocytosis is a major target for murid herpesvirus-4 neutralization

2012 ◽  
Vol 93 (6) ◽  
pp. 1316-1327 ◽  
Author(s):  
Daniel L. Glauser ◽  
Laurent Gillet ◽  
Philip G. Stevenson
Keyword(s):  
Protein Conformation ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Antibody Responses ◽  
Cell Binding ◽  
Late Endosomes ◽  
Glycoprotein H ◽  
Dependent Protein ◽  
Herpesvirus 4 ◽  
Major Target

Herpesviruses consistently transmit from immunocompetent carriers, implying that their neutralization is hard to achieve. Murid herpesvirus-4 (MuHV-4) exploits host IgG Fc receptors to bypass blocks to cell binding, and pH-dependent protein conformation changes to unveil its fusion machinery only after endocytosis. Nevertheless, neutralization remains possible by targeting the virion glycoprotein H (gH)–gL heterodimer, and the neutralizing antibody responses of MuHV-4 carriers are improved by boosting with recombinant gH–gL. We analysed here how gH–gL-directed neutralization works. The MuHV-4 gH–gL binds to heparan sulfate. However, most gH–gL-specific neutralizing antibodies did not block this interaction; neither did they act directly on fusion. Instead, they blocked virion endocytosis and transport to the late endosomes, where membrane fusion normally occurs. The poor endocytosis of gH–gL-neutralized virions was recapitulated precisely by virions genetically lacking gL. Therefore, driving virion uptake appears to be an important function of gH–gL that provides a major target for antibody-mediated neutralization.

scholarly journals Recruitment of Mammalian Cell Fibronectin to the Surface of Chlamydia trachomatis

2002 ◽  
Vol 70 (7) ◽  
pp. 3935-3938 ◽  
Author(s):  
Betsy J. Kleba ◽  
Erin Banta ◽  
Erika A. Lindquist ◽  
Richard S. Stephens
Keyword(s):  
Chlamydia Trachomatis ◽  
Heparan Sulfate ◽  
Host Cell ◽  
Mammalian Cell ◽  
Pathogenic Bacteria ◽  
Cell Binding ◽  
Cell Functions ◽  
Wide Range

ABSTRACT Pathogenic bacteria exploit the presence of various host cell molecules in order to colonize new tissues. Fibronectin is involved in a wide range of cell functions in vivo, and staphylococci, streptococci, and gonococci have evolved mechanisms to utilize this glycoprotein to mediate host cell binding. We show that elementary bodies (EB) from two biovars of Chlamydia trachomatis recruit fibronectin to their surfaces upon lysis of the host cell. We also demonstrate that a heparan sulfate lyase-sensitive molecule on chlamydial EB is responsible for binding at least a portion of this fibronectin.

scholarly journals Membrane fusion process of Semliki Forest virus. II: Cleavage-dependent reorganization of the spike protein complex controls virus entry.

1992 ◽  
Vol 116 (2) ◽  
pp. 349-357 ◽  
Author(s):  
A Salminen ◽  
J M Wahlberg ◽  
M Lobigs ◽  
P Liljeström ◽  
H Garoff
Keyword(s):  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Wild Type Virus ◽  
Wild Type ◽  
Cell Binding ◽  
Virus Budding ◽  
Virus Particles ◽  
Acidic Conditions ◽  
Ph Conditions ◽  
Heterodimeric Complex

The envelope of the Semliki Forest virus (SFV) contains two transmembrane proteins, E2 and E1, in a heterodimeric complex. The E2 subunit is initially synthesized as a precursor protein p62, which is proteolytically processed to the mature E2 form before virus budding at the plasma membrane. The p62 (E2) protein mediates binding of the heterodimer to the nucleocapsid during virus budding, whereas E1 carries the entry functions of the virus, that is, cell binding and low pH-mediated membrane fusion activity. We have investigated the significance of the cleavage event for the maturation and entry of the virus. To express SFV with an uncleaved p62 phenotype, BHK-21 cells were transfected by electroporation with infectious viral RNA transcribed from a full-length SFV cDNA clone in which the p62 cleavage site had been changed. The uncleaved p62E1 heterodimer was found to be used for the formation of virus particles with an efficiency comparable to the wild type E2E1 form. However, in contrast to the wild type virus, the mutant virus was virtually noninfectious. Noninfectivity resulted from impaired uptake into cells, as well as from the inability of the virus to promote membrane fusion in the mildly acidic conditions of the endosome. This inability could be reversed by mild trypsin treatment, which converted the viral p62E1 form into the mature E2E1 form, or by treating the virus with a pH 4.5 wash, which in contrast to the more mild pH conditions of endosomes, effectively disrupted the p62E1 subunit association. We conclude that the p62 cleavage is not needed for virus budding, but regulates entry functions of the E1 subunit by controlling the heterodimer stability in acidic conditions.

scholarly journals Surface-exposed Amino Acid Residues of HPV16 L1 Protein Mediating Interaction with Cell Surface Heparan Sulfate

2007 ◽  
Vol 282 (38) ◽  
pp. 27913-27922 ◽  
Author(s):  
Maren Knappe ◽  
Sabrina Bodevin ◽  
Hans-Christoph Selinka ◽  
Dorothe Spillmann ◽  
Rolf E. Streeck ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Capsid Protein ◽  
Mutational Analysis ◽  
Cell Attachment ◽  
Human Papillomaviruses ◽  
Amino Acid Residues ◽  
Cell Binding ◽  
Lysine Residues

Efficient infection of cells by human papillomaviruses (HPVs) and pseudovirions requires primary interaction with cell surface proteoglycans with apparent preference for species carrying heparan sulfate (HS) side chains. To identify residues contributing to virus/cell interaction, we performed point mutational analysis of the HPV16 major capsid protein, L1, targeting surface-exposed amino acid residues. Replacement of lysine residues 278, 356, or 361 for alanine reduced cell binding and infectivity of pseudovirions. Various combinations of these amino acid exchanges further decreased cell attachment and infectivity with residual infectivity of less than 5% for the triple mutant, suggesting that these lysine residues cooperate in HS binding. Single, double, or triple exchanges for arginine did not impair infectivity, demonstrating that interaction is dependent on charge distribution rather than sequence-specific. The lysine residues are located within a pocket on the capsomere surface, which was previously proposed as the putative receptor binding site. Fab fragments of binding-neutralizing antibody H16.56E that recognize an epitope directly adjacent to lysine residues strongly reduced HS-mediated cell binding, further corroborating our findings. In contrast, mutation of basic surface residues located in the cleft between capsomeres outside this pocket did not significantly reduce interaction with HS or resulted in assembly-deficient proteins. Computer-simulated heparin docking suggested that all three lysine residues can form hydrogen bonds with 2-O-, 6-O-, and N-sulfate groups of a single HS molecule with a minimal saccharide domain length of eight monomer units. This prediction was experimentally confirmed in binding experiments using capsid protein, heparin molecules of defined length, and sulfate group modifications.

scholarly journals Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion

2020 ◽  
Vol 117 (50) ◽  
pp. 32105-32113 ◽  
Author(s):  
Ruochen Zang ◽  
James Brett Case ◽  
Eylan Yutuc ◽  
Xiucui Ma ◽  
Sheng Shen ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Vesicular Stomatitis Virus ◽  
Molecular Basis ◽  
Enveloped Viruses ◽  
Late Endosomes ◽  
Wide Range ◽  
Therapeutic Development ◽  
Antiviral Activities ◽  
Vesicular Stomatitis ◽  
Catalyzed Membrane

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.

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