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 RSV Vaccine Based on Rhabdoviral Vector Protects after Single Immunization

Vaccines ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 59 ◽  
Author(s):  
Wilmschen ◽  
Schneider ◽  
Peters ◽  
Bayer ◽  
Issmail ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Lymphocytic Choriomeningitis ◽  
Effective Vaccine ◽  
Vector Vaccine ◽  
Viral Loads ◽  
Glycoprotein G ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Syncytial Virus ◽  
Tract Infections

The respiratory syncytial virus (RSV) is one major cause of lower respiratory tract infections in childhood and an effective vaccine is still not available. We previously described a new rhabdoviral vector vaccine, VSV-GP, a variant of the vesicular stomatitis virus (VSV), where the VSV glycoprotein G is exchanged by the glycoprotein GP of the lymphocytic choriomeningitis virus. Here, we evaluated VSV-GP as vaccine vector for RSV with the aim to induce RSV neutralizing antibodies. Wild-type F (Fwt) or a codon optimized version (Fsyn) were introduced at position 5 into the VSV-GP genome. Both F versions were efficiently expressed in VSV-GP-F infected cells and incorporated into VSV-GP particles. In mice, high titers of RSV neutralizing antibodies were induced already after prime and subsequently boosted by a second immunization. After challenge with RSV, viral loads in the lungs of immunized mice were reduced by 2–3 logs with no signs of an enhanced disease induced by the vaccination. Even a single intranasal immunization significantly reduced viral load by a factor of more than 100-fold. RSV neutralizing antibodies were long lasting and mice were still protected when challenged 20 weeks after the boost. Therefore, VSV-GP is a promising candidate for an effective RSV vaccine.

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 Deficient Incorporation of Rabies Virus Glycoprotein into Virions Enhances Virus-Induced Immune Evasion and Viral Pathogenicity

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 218 ◽  
Author(s):  
Chunfu Li ◽  
Hongliang Zhang ◽  
Lina Ji ◽  
Xiao Wang ◽  
Yongjun Wen ◽  
...  
Keyword(s):  
Immune Response ◽  
Rabies Virus ◽  
Recombinant Virus ◽  
Neutralizing Antibodies ◽  
Single Copy ◽  
Wild Type ◽  
Antiviral Immune Response ◽  
Glycoprotein G ◽  
Lethal Infection ◽  
Infected Cells

Previous studies have shown that wild-type (wt) rabies virus (RABV) evades the host immune response by restricting expression of glycoprotein (G), which blocks activation of dendritic cells (DCs) and induces production of virus-neutralizing antibodies (VNAs). In the present study, wt RABVs not only restricted G expression but also reduced incorporation of G into mature virions compared with laboratory-adapted viruses. A recombinant RABV expressing triple G was used to further determine whether G expression relates to incorporation. The recombinant virus showed higher expression and incorporation of G and activated more DCs than the virus that expressed a single copy of G. Removal of G from viruses using subtilisin or Dithiothreitol (DTT)/ Nonidet P-40 (NP40) almost completely abolishes DC activation and VNA production. Consequently, these G-depleted viruses cause lethal infection in mice. Thus, wt RABVs can subvert DC-induced antiviral immune response and maintain pathogenicity by decreasing G expression in infected cells and G incorporation into virions.

scholarly journals Safe and effective two-in-one replicon-and-VLP minispike vaccine for COVID-19: Protection of mice after a single immunization

2021 ◽  
Vol 17 (4) ◽  
pp. e1009064
Author(s):  
Alexandru A. Hennrich ◽  
Bevan Sawatsky ◽  
Rosalía Santos-Mandujano ◽  
Dominic H. Banda ◽  
Martina Oberhuber ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Antigen Expression ◽  
Vaccine Platform ◽  
Dna And Rna ◽  
Glycoprotein G ◽  
Virus Like Particle ◽  
Main Target ◽  
Boost Immunization ◽  
Vesicular Stomatitis ◽  
Anchor Sequence

Vaccines of outstanding efficiency, safety, and public acceptance are needed to halt the current SARS-CoV-2 pandemic. Concerns include potential side effects caused by the antigen itself and safety of viral DNA and RNA delivery vectors. The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which might cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine complemented with VSV G, VSVΔG-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients, and protected transgenic K18-hACE2 mice from COVID-19-like disease. Homologous boost immunization further enhanced virus neutralizing activity. The results demonstrate that non-spreading rhabdovirus RNA replicons expressing minispike proteins represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen.

scholarly journals Binding of viral glycoprotein mRNA to endoplasmic reticulum membranes is disrupted by puromycin.

1977 ◽  
Vol 74 (2) ◽  
pp. 358-364 ◽  
Author(s):  
H F Lodish ◽  
S Froshauer
Keyword(s):  
Endoplasmic Reticulum ◽  
Ionic Strength ◽  
Vesicular Stomatitis Virus ◽  
Direct Interaction ◽  
Viral Glycoprotein ◽  
Glycoprotein G ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Low Ionic Strength ◽  
Er Membrane

Previous studies showed that the glycoprotein (G) of vesicular stomatitis virus is synthesized in association with the endoplasmic reticulum (ER) membrane and that all G mRNA co-fractionates with ER membrane. Here, we show that treatment of infected cells with puromycin results in dissociation of G mRNA, and presumably the associated ribosomes, from the ER membrane. Even it extracts from treated cells are kept at low ionic strength (0.01 M KCl), over 80% of G mRNA is found unattached to membranes. There is no evidence for direct interaction of GmRNA with membranes; rather, the linkage apparently is mediated by the nascent G polypeptide.

scholarly journals Multiple Pathways Involved in Porcine Parvovirus Cellular Entry and Trafficking toward the Nucleus

2010 ◽  
Vol 84 (15) ◽  
pp. 7782-7792 ◽  
Author(s):  
Maude Boisvert ◽  
Sandra Fernandes ◽  
Peter Tijssen
Keyword(s):  
Sialic Acids ◽  
Porcine Parvovirus ◽  
Actin Network ◽  
Cellular Mechanisms ◽  
Microtubule Network ◽  
Late Endosomes ◽  
Chemical Inhibitors ◽  
Endosomal Acidification ◽  
Infected Cells ◽  
Gain Access

ABSTRACT Porcine parvovirus (PPV) is a major cause of reproductive failure in swine. The mechanisms implicated in the first steps of infection that lead to the delivery of the PPV genome to the nucleus are poorly understood. In the present work, a panel of chemical inhibitors was used to dissect the cellular mechanisms involved in establishing a PPV infection. The results demonstrated that following binding to sialic acids on cell surface glycoproteins, the virus used both clathrin-mediated endocytosis and macropinocytosis pathways to gain access into cells. Virus obtained from infected cells was present either as isolated particles or as aggregates, and these two forms could be separated by low-speed centrifugation. Isolated and purified particles strongly preferred entry by clathrin-mediated endocytosis, whereas aggregates clearly favored macropinocytosis. Subsequent endosomal acidification and traffic to the late endosomes were also shown to be essential for infection. The microtubule network was found to be important during the first 10 h of infection, whereas an intact actin network was required for almost the whole viral cycle. Proteasome processing was found to be essential, and capsid proteins were ubiquitinated relatively early during infection. Taken together, these results provided new insights into the first steps of PPV infection, including the use of alternative entry pathways, unique among members of this viral family.

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 OvHV-2 Glycoprotein B Delivered by a Recombinant BoHV-4 Is Immunogenic and Induces Partial Protection against Sheep-Associated Malignant Catarrhal Fever in a Rabbit Model

Vaccines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 90
Author(s):  
Smriti Shringi ◽  
Donal O’Toole ◽  
Emily Cole ◽  
Katherine N. Baker ◽  
Stephen N. White ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Lethal Dose ◽  
Rabbit Model ◽  
Genetically Engineered ◽  
Glycoprotein B ◽  
Malignant Catarrhal Fever ◽  
Efficacious Vaccine ◽  
Herpesvirus 4

An efficacious vaccine for sheep-associated malignant catarrhal fever (SA-MCF) is important for the livestock industry. Research towards SA-MCF vaccine development is hindered by the absence of culture systems to propagate the causative agent, ovine herpesvirus-2 (OvHV-2), which means its genome cannot be experimentally modified to generate an attenuated vaccine strain. Alternative approaches for vaccine development are needed to deliver OvHV-2 antigens. Bovine herpesvirus 4 (BoHV-4) has been evaluated as a vaccine vector for several viral antigens with promising results. In this study, we genetically engineered BoHV-4 to express OvHV-2 glycoprotein B (gB) and evaluated its efficacy as an SA-MCF vaccine using a rabbit model. The construction of a viable recombinant virus (BoHV-4-AΔTK-OvHV-2-gB) and confirmation of OvHV-2 gB expression were performed in vitro. The immunization of rabbits with BoHV-4-AΔTK-OvHV-2-gB elicited strong humoral responses to OvHV-2 gB, including neutralizing antibodies. Following intra-nasal challenge with a lethal dose of OvHV-2, 42.9% of the OvHV-2 gB vaccinated rabbits were protected against SA-MCF, while all rabbits in the mock-vaccinated group succumbed to SA-MCF. Overall, OvHV-2 gB delivered by the recombinant BoHV-4 was immunogenic and partly protective against SA-MCF in rabbits. These are promising results towards an SA-MCF vaccine; however, improvements are needed to increase protection rates.

scholarly journals Safe and effective two-in-one replicon-and-VLP minispike vaccine for COVID-19

2020 ◽  
Author(s):  
Alexandru A. Hennrich ◽  
Dominic H. Banda ◽  
Martina Oberhuber ◽  
Anika Schopf ◽  
Verena Pfaffinger ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Neutralizing Antibodies ◽  
Antigen Expression ◽  
List Type ◽  
Vaccine Platform ◽  
Glycoprotein G ◽  
Virus Like Particle ◽  
Boost Immunization ◽  
Vesicular Stomatitis ◽  
Anchor Sequence

SummaryThe large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which may cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine, VSVΔG-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients. Boost immunization with the identical replicon further enhanced neutralizing activity. These results demonstrate that rhabdovirus minispike replicons represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen.HighlightsSARS-CoV-2 S RBD antigen is preferred over entire S to preclude potential disease enhancing antibodiesconstruction of a chimeric rhabdovirus minispike protein presenting RBD in native conformationconstruction of single round VSV and rabies virus replicon vaccinespresentation of minispike antigen on cells and on noninfectious VLPsstrong induction of SARS-CoV-2 neutralizing antibodies by the VSV replicon/VLP system in vaccinated mice

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