Shedding of Gs Protein (A Soluble Form of the Viral Glycoprotein) by the Rabies Virus-Infected BHK-21 Cells

Virology ◽  
1993 ◽  
Vol 195 (2) ◽  
pp. 541-549 ◽  
Author(s):  
Kinjiro Morimoto ◽  
Yasumasa Iwatani ◽  
Akihiko Kawai
Keyword(s):  
Rabies Virus ◽  
Protein A ◽  
Soluble Form ◽  
Viral Glycoprotein ◽  
Gs Protein

scholarly journals Mutations Conferring Resistance to Neutralization by a Soluble Form of the Neurotrophin Receptor (p75NTR) Map outside of the Known Antigenic Sites of the Rabies Virus Glycoprotein

2002 ◽  
Vol 76 (21) ◽  
pp. 10756-10765 ◽  
Author(s):  
Christelle Langevin ◽  
Christine Tuffereau
Keyword(s):  
Rabies Virus ◽  
Protein A ◽  
Neurotrophin Receptor ◽  
Soluble Form ◽  
Single Mutation ◽  
Soluble Receptor ◽  
Fc Fragment ◽  
Glycoprotein Gene ◽  
Antigenic Sites ◽  
Resistant Mutants

ABSTRACT The neurotrophin receptor (p75NTR) serves as a receptor for rabies virus (RV). We expressed and purified a soluble chimera consisting of the p75NTR ectodomain fused to the human immunoglobulin G1 (IgG1) Fc fragment (p75-Fc). Although p75-Fc interacts with RV, the infectivity of RV did not decrease significantly when it was incubated in the presence of the soluble receptor alone. However, when it was subsequently incubated with an antihuman IgG directed against the Fc fragment of p75-Fc, the infectivity of RV was significantly lowered (>90%), whereas incubation with antihuman IgG alone had no effect. We then selected eight independent RV mutants that were not neutralized by p75-Fc and antihuman IgG (srr [soluble receptor resistant] mutants). Each mutant carried a single mutation in the glycoprotein gene leading to one amino acid substitution in the protein. A total of four different substitutions were found. Two of the mutations were located at position 318 (phenylalanine replaced by a serine or a valine residue), and two were located at position 352 (histidine replaced by a tyrosine or an arginine residue). All of the mutations prevented the interaction with p75NTR as either a soluble or a membrane-anchored form. Two mutants (F318S) and (H352R) resulted in the formation of small plaques on BSR cells, probably due to the slower maturation of the glycoprotein. Immunoprecipitation, immunofluorescence, and neutralization assays showed that the four mutated glycoproteins still interacted with representative anti-RV glycoprotein monoclonal antibodies (MAbs), indicating that p75NTR binds outside of the known RV glycoprotein antigenic sites.

Detection of Rabies Virus and Its Antibodies Using Staphylococcus aureus Protein A

2015 ◽  
Vol 43 (1) ◽  
pp. 87-93
Author(s):  
Hemmat A ◽  
A Albehwar ◽  
M Shendy
Keyword(s):  
Staphylococcus Aureus ◽  
Rabies Virus ◽  
Protein A

A new recombinant rabies virus expressing a green fluorescent protein: A novel and fast approach to quantify virus neutralizing antibodies

Biologicals ◽  
2019 ◽  
Vol 59 ◽  
pp. 56-61 ◽  
Author(s):  
Shuyun Qin ◽  
Dmitriy Volokhov ◽  
Elvira Rodionova ◽  
Christoph Wirblich ◽  
Matthias J. Schnell ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Rabies Virus ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Protein A ◽  
Fast Approach ◽  
Green Fluorescent

scholarly journals Expression and Solubilization of Insect Cell-Based Rabies Virus Glycoprotein and Assessment of Its Immunogenicity and Protective Efficacy in Mice

2011 ◽  
Vol 18 (10) ◽  
pp. 1673-1679 ◽  
Author(s):  
R. Ramya ◽  
B. Mohana Subramanian ◽  
V. Sivakumar ◽  
R. L. Senthilkumar ◽  
K. R. S. Sambasiva Rao ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Insect Cell ◽  
Neutralizing Antibodies ◽  
Subunit Vaccine ◽  
Vaccine Development ◽  
Protective Efficacy ◽  
Vector System ◽  
Soluble Form ◽  
Rabies Virus Glycoprotein ◽  
Virus Glycoprotein

ABSTRACTRabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence. The glycoprotein (G) was cloned using the baculovirus expression vector system (BEVS) and expressed inSpodoptera frugiperda(Sf-9) cells. In order to obtain a soluble form of G suitable for experimentation in mice, 18 different combinations of buffers and detergents were evaluated for their ability to solubilize the insect cell membrane-associated G. The combination that involved 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) detergent in lysis buffer 1, formulated with Tris, NaCl, 10% dimethyl sulfoxide (DMSO), and EDTA, gave the highest yield of soluble G, as evidenced by the experimental data. Subsequently, several other parameters, such as the concentration of CHAPS and the duration and temperature of the treatment for the effective solubilization of G, were optimized. The CHAPS detergent, buffered at a concentration of 0.4% to 0.7% (wt/vol) at room temperature (23 to 25°C) for 30 min to 1 h using buffer 1, containing 10% DMSO, resulted in consistently high yields. The G solubilized using CHAPS detergent was found to be immunogenic when tested in mice, as evidenced by high virus-neutralizing antibody titers in sera and 100% protection upon virulent intracerebral challenge with the challenge virus standard (CVS) strain of rabies virus. The results of the mice study indicated that G solubilized with CHAPS detergent retained the immunologically relevant domains in the native conformation, thereby paving the way for producing a cell-free and efficacious subunit vaccine.

scholarly journals Rabies Virus-Induced Membrane Fusion Pathway

2000 ◽  
Vol 150 (3) ◽  
pp. 601-612 ◽  
Author(s):  
Yves Gaudin
Keyword(s):  
Lipid Bilayers ◽  
Rabies Virus ◽  
Fusion Process ◽  
Fusion Pore ◽  
Viral Glycoprotein ◽  
Rate Limiting Step ◽  
Glycoprotein G ◽  
Lipid Organization ◽  
Target Membrane ◽  
Peptide Insertion

Fusion of rabies virus with membranes is triggered at low pH and is mediated by the viral glycoprotein (G). The rabies virus-induced fusion pathway was studied by investigating the effects of exogenous lipids having various dynamic molecular shapes on the fusion process. Inverted cone-shaped lysophosphatidylcholines (LPCs) blocked fusion at a stage subsequent to fusion peptide insertion into the target membrane. Consistent with the stalk-hypothesis, LPC with shorter alkyl chains inhibited fusion at lower membrane concentrations and this inhibition was compensated by the presence of oleic acid. However, under suboptimal fusion conditions, short chain LPCs, which were translocated in the inner leaflet of the membranes, considerably reduced the lag time preceding membrane merging, resulting in faster kinetics of fusion. This indicated that the rate limiting step for fusion is the formation of a fusion pore in a diaphragm of restricted hemifusion. The previously described cold-stabilized prefusion complex was also characterized. This intermediate is at a well-advanced stage of the fusion process when the hemifusion diaphragm is destabilized, but lipid mixing is still restricted, probably by a ring-like complex of glycoproteins. I provide evidence that this state has a dynamic character and that its lipid organization can reverse back to two lipid bilayers.

scholarly journals Trypanosoma cruzi trans-sialidase: enhancement of virulence in a murine model of Chagas' disease.

1995 ◽  
Vol 181 (5) ◽  
pp. 1693-1703 ◽  
Author(s):  
M Chuenkova ◽  
M E Pereira
Keyword(s):  
Trypanosoma Cruzi ◽  
Substrate Specificity ◽  
Chagas Disease ◽  
Severe Combined Immunodeficiency ◽  
Protein A ◽  
Inflammatory Cells ◽  
Soluble Form ◽  
Maximum Enhancement

Trypanosoma cruzi, the etiological agent of Chagas' disease, expresses a trans-sialidase at highest levels in infective trypomastigotes, where it attaches to the plasma membrane by a glycophosphoinositol linkage. Bound enzyme sheds into the extracellular milieu in a soluble form. Experiments performed in vitro suggest that the trans-sialidase participates in several parameters of T. cruzi-host interactions, like cell adhesion and complement resistance. However, the role that membrane-bound and soluble trans-sialidase plays in the infection of mammals is not understood. To begin to study the role the enzyme may play in vivo, T. cruzi trypomastigotes were inoculated subcutaneously into mice that had been sensitized for various times with the purified protein. A single dose of either endogenous or recombinant trans-sialidase injected into the connective tissues of BALB/c mice greatly enhanced parasitemia and mortality. Maximum enhancement was achieved with 1-2-h priming. Injection of the enzyme after the parasites had been established in the inoculation site had little, if any, consequence in modifying virulence. The enhancement did not seem to be through a direct effect of the enzyme on trypomastigote-host cell interactions because it occurred when the sites of trans-sialidase sensitization and parasite inoculation were physically separate. Rather, virulence enhancement seemed to depend on inflammatory cells, since priming with trans-sialidase had no significant effect in severe combined immunodeficiency mice, which lack functional T and B lymphocytes. However, antibody response to T. cruzi in the trans-sialidase-primed BALB/c mice was the same as in the control animals. Virulence enhancement was specific for the trans-sialidase because it did not occur in mice primed with Newcastle virus sialidase, which has the same substrate specificity as the T. cruzi enzyme, or with the sialidase from the bacterium Vibrio cholerae, whose substrate specificity is broader than the trypanosome sialidase. Furthermore, no enhancement of virulence occurred after sensitization with another adhesion protein (penetrin) purified from T. cruzi trypomastigotes and engineered bacteria, nor with bacterial lipopolysaccharide. The virulence-promoting activity of soluble trans-sialidase in the mouse model may be physiologically relevant because it was achieved with tiny doses, approximately 1-2 microgram/kg, raising the possibility that neutralization of the enzyme with specific probes could impair the development of Chagas' disease. In fact, a monoclonal antibody specific for the tandem repeat in the trans-sialidase COOH terminus enhanced infection of BALB/c mice, in agreement with earlier experiments in vitro, whereas antibodies against an amino acid sequence in the Cys region had the opposite effect.

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